RNA vaccine against SARS-CoV-2 variants

ABSTRACT

The present invention is directed to a nucleic acid suitable for use in treatment or prophylaxis of an infection with a coronavirus, preferably with a Coronavirus SARS-CoV-2, or a disorder related to such an infection, preferably COVID-19. The present invention is also directed to compositions, polypeptides, and vaccines. The compositions and vaccines preferably comprise at least one of said nucleic acid sequences, preferably nucleic acid sequences in association a lipid nanoparticle (LNP). The invention is also directed to first and second medical uses of the nucleic acid, the composition, the polypeptide, the combination, the vaccine, and the kit, and to methods of treating or preventing a coronavirus infection, preferably a Coronavirus infection.

The present application claims priority to U.S. provisional applicationNo. 63/129,395 filed on Dec. 22, 2020; PCT application No.PCT/EP2021/052455 filed on Feb. 3, 2021; PCT application No.PCT/EP2021/069626 filed on Jul. 14, 2021 and PCT application No.PCT/EP2021/069632 filed Jul. 14, 2021. Each of which of theaforementioned applications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention is inter alia directed to an RNA suitable for usein treatment or prophylaxis of an infection with SARS-CoV-2 variants,including, but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron,South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2,BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1(Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India),AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351(Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil),B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria),B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda),B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil),C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu,Columbia), or a disorder related to such infections. The presentinvention also concerns compositions, polypeptides, and vaccines. Thecompositions and vaccines preferably comprise at least one of said RNAsequences, preferably RNA in association with lipid nanoparticles(LNPs).

List

The invention is also directed to first and second medical uses of theRNA, the composition, the vaccine, and the kit, and to methods oftreating or preventing a SARS-CoV-2 infection caused by SARS-Cov-2variants, including, but not limited to: C.1.2 (South Africa), B.1.1.529(Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2,BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619(Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India),AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3(India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma,Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta,Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1(Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta,Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621(Mu, Columbia).

Coronaviruses are highly contagious, enveloped, positive single strandedzoonotic RNA viruses of the Coronaviridae family.

Coronaviruses are genetically highly variable, and individual virusspecies have the potential to infect several host species by overcomingthe species barrier. In late 2019, an outbreak of respiratory diseasecaused by a novel Coronavirus strain was reported in Wuhan City, HubeiProvince, China. The novel Coronavirus was named “severe acuterespiratory syndrome coronavirus 2” (SARS-CoV-2). Typical symptoms of aSARS-CoV-2 caused virus infection, also referred to as COVID-19 disease,include fever, cough, shortness of breath, and pneumonia, with highmortality rates in the elderly population. In March 2020, the WHOdeclared the SARS-CoV-2 outbreak a pandemic. In addition, someindividuals suffer the effects of COVID-19 infection for weeks to monthsafter infection. This population is referred to “long Covid”. Commonsigns and symptoms that linger over time include: fatigue, shortness ofbreath or difficulty breathing, cough, joint pain, chest pain, memory,concentration or sleep problems, muscle pain or headache, fast orpounding heartbeat, loss of smell or taste, depression or anxiety,fever, dizziness on standing, worsened symptoms after physical or mentalactivities.

During the pandemic, new SARS-CoV-2 variant strains were emerging thatare often more contagious or more pathogenic than the originalSARS-CoV-2 strain. Such new emerging SARS-CoV-2 strains may potentiallylead to a reduced efficiency of first-generation vaccines that weredeveloped against the original SARS-CoV-2 strain. Further, it is unclearwhether a boost vaccination with a vaccine specifically designed againstnew emerging SARS-CoV-2 strains in subjects which have been vaccinatedagainst the original SARS-CoV-2 strain will lead to protective immuneresponses against the new emerging SARS-CoV-2 strains.

SUMMARY OF THE INVENTION

Therefore, it is one object of the underlying invention to provide anRNA-based vaccine for SARS-CoV-2 infections, in particular SARS-CoV-2infections caused by novel emerging SARS-CoV-2 variant strains. Suchnovel emerging strains include but not limited to: C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia). RNA based vaccination represents one of the mostpromising techniques for new vaccines against new emerging SARS-CoV-2viruses. RNA can be genetically engineered and adapted to new emergingSARS-CoV-2 strains and administered to a human subject, wheretransfected cells directly produce the encoded antigen provided by theRNA which results in immunological responses.

As further defined in the claims and the underlying description, theseobjects are inter alia solved by providing an RNA comprising at leastone coding sequence encoding at least one antigenic peptide or proteinderived from SARS-CoV-2, e.g. comprising at least one mutation derivedfrom a SARS-Cov-2 strain including, but not limited to: C.1.2 (SouthAfrica), B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia).

In a preferable embodiment of the invention the RNA and RNA-basedvaccine comprises an RNA encoding at least one antigenic peptide derivedfrom a SARS-CoV-2 spike protein, e.g. comprising a spike protein fromderived from a SARS-Cov-2 strain including, but not limited to: C.1.2(South Africa), B.1.1.529 (Omicron, South Africa) (including BA.1_v1,BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3(Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada),AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus,India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha,UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US),B.1.525 (Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, NewYork, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta,India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta,Philippines), and/or B.1.621 (Mu, Columbia)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows significant IgG1 and IgG2a binding antibody responses tothe receptor binding domain (RBD) of ancestral SARS-CoV-2 and the RBD ofthe B.1.351 variant on day 14 (FIG. 1A-D) and on day 21 (FIG. 1E-H) forthe group vaccinated with the mRNA vaccine CV2CoV and the CV2CoV.351,both formulated in LNPs. On day 14, FIG. 1A shows comparable IgG1response for all groups (ancestral SARS-CoV-2 receptor binding domain(RBD) protein coating) and FIG. 1B shows comparable IgG2a titers for allvaccination designs (ancestral RBD protein coating). On day 14, FIG. 1Cshows comparable IgG1 response for all vaccination designs (RBD B.1.351variant K417N, E484K, N501Y protein coating) and FIG. 1D showscomparable IgG2a titers for all vaccination designs (RBD B.1.351 variantK417N, E484K, N501Y protein coating). On day 21, FIG. 1E showscomparable IgG1 response for all vaccination designs (ancestralSARS-CoV-2 receptor binding domain (RBD) protein coating) and FIG. 1Fshows comparable IgG2a titers for all vaccination designs (ancestralSARS-CoV-2 receptor binding domain (RBD) protein coating). On day 21,FIG. 1G shows comparable IgG1 response for all vaccination designs (RBDB.1.351 variant K417N, E484K, N501Y protein coating) and FIG. 1H showscomparable IgG2a titers for all vaccination designs (RBD B.1.351 variantK417N, E484K, N501Y protein coating).

FIG. 2 shows significant induction of virus neutralizing titers (VNTs)assessed in a cytopathic effect (CPE)-based assay using ancestralSARS-CoV-2. FIG. 2A on day 14 and FIG. 2B on day 21 show an increase ofthe VNT for all groups B-H. Co-delivery of the mRNA vaccine CV2CoV andthe CV2CoV.351 into the same leg (group F and G) or different legs(group H) can generate responses against both vaccine variants on day 14and day 21. FIG. 2C on day 42 shows an increased level of VNT for allgroups (group B-H). Co-delivery of both vaccine variants into the sameleg (group F and G) or into different legs (group H) can generateresponses against both variants on day 42.

FIG. 3 shows significant induction of VNTs) assessed in a CPE-basedassay using B.1.351 variant SARS-CoV-2. FIG. 3A on day 14 and FIG. 3B onday 21 show an increase of the VNT for all groups B-H. Co-delivery ofthe mRNA vaccine CV2CoV and the CV2CoV.351 into the same leg (group Fand G) or different legs (group H) can generate responses against bothvaccine variants on day 14 and day 21. FIG. 3C on day 42 shows anincreased level of VNT for all groups (group B-H). Co-delivery of bothvaccine variants into the same leg (group F and G) or into differentlegs (group H) can generate responses against both variants on day 42.

FIG. 4 shows significant induction of VNTs assessed in a CPE-based assayusing B.1.1.7 variant SARS-CoV-2 (FIG. 4A) or P.1 (B.1.1.28) (FIG. 4B).FIG. 4A on day 42 shows an increased level of VNT using B.1.1.7 variantfor all groups (group B-H). Co-delivery of both vaccine variants intothe same leg (group F and G) or into different legs (group H) cangenerate responses against both variants on day 42. FIG. 4B on day 42shows an increased level of VNT using B.1.1.28 P.1 variant for allgroups (group B-H). Co-delivery of both vaccine variants into the sameleg (group F and G) or into different legs (group H) can generateresponses against both variants on day 42.

FIG. 5 shows significant IgG1 and IgG2a binding antibody responses onday 14 (FIG. 5A-D) and on day 21 (FIG. 5E-H) for the groups vaccinatedwith CV2CoV and CV2CoV.351. On day 14, FIG. 5A (IgG1 titer) and 5B(IgG2a titer) show dose-dependent levels of binding antibody titersusing doses of 0.5 μg, 2 μg, 8 μg and 40 μg and reached saturation inthe groups vaccinated with 40 μg (ancestral SARS-CoV-2 receptor bindingdomain (RBD) protein coating). On day 14, FIG. 5C (IgG1 titer) and 5D(IgG2a titer) show dose-dependent levels of binding antibody titersusing doses of 0.5 μg, 2 μg, 8 μg and 40 μg and reached saturation ingroups vaccinated with 40 μg (RBD B.1.351 variant K417N, E484K, N501Yprotein coating). On day 21, FIG. 5E (IgG1 titer) and 5F (IgG2a titer)show a dose-dependent levels of binding antibody titer and saturatedIgG1 titer and IgG2a titer for the CV2CoV.351 vaccination for allamounts and a saturation for amounts >8 μg (group I) for CV2CoV(ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating). Onday 21, FIG. 5G (IgG1 titer) and 5H (IgG2a titer) showdose-dependentlevels of binding antibody titer and saturated IgG1 and IgG2a titersupon vaccination with CV2CoV.351 for all amounts and a saturation foramounts >8 μg (group I) for the mRNA vaccine the CV2CoV (RBD B.1.351variant K417N, E484K, N501Y protein coating).

FIG. 6 shows significant induction of VNTs assessed in a CPE-based assayusing ancestral SARS-CoV-2 (CV2CoV, FIGS. 6A, 6C and 6E) or B.1.351variant SARS-CoV-2 (CV2CoV.351, FIGS. 6B, 6D and 6F) on day 14, day 21and day 42. FIG. 6 also shows significant induction of VNTs assessed ina CPE-based using B.1.1.7 variant SARS-CoV-2 (FIG. 6G) or B.1.1.28 P.1variant (FIG. 6H) on day 42. FIG. 6A shows that the B.1.351 variantvaccine CV2CoV.351 induces dose-dependent VNTs against ancestralSARS-CoV-2 (heterologous response) on day 14 in all dose groups.Compared to responses upon vaccination with CV2CoV (homologousresponse), VNTs in CV2CoV.351 vaccinated groups are decreased by afactor of approx. 2 on day 14. FIG. 6B shows that CV2CoV.351 inducesdose-dependent VNTs against B.1.351 SARS-CoV-2 (homologous response) onday 14 in all dose groups. CV2CoV.351 vaccination elicited high levelsof VNTs against homologous virus that were 45× increased on day 14,compared to heterologous VNTs against ancestral virus (averagedifference of all dose groups). In comparison to vaccination withCV2CoV, VNTs induced by CV2CoV.351 were increased by a factor of 41 onday 14 (average difference of all dose groups). FIG. 6C shows that theB.1.351 variant vaccine CV2CoV.351 induces dose-dependent VNTs againstancestral SARS-CoV-2 (heterologous response) on day 21 in all dosegroups. Compared to responses upon vaccination with CV2CoV (homologousresponse), VNTs in CV2CoV.351 vaccinated groups are decreased by afactor of approx. 2 on day 21. FIG. 6D shows that CV2CoV.351 inducesslightly dose-dependent VNTs against B.1.351 SARS-CoV-2 (homologousresponse) on day 21 in all dose groups. CV2CoV.351 vaccination elicitedhigh levels of VNTs against homologous virus that were 35× increased onday 21, compared to heterologous VNTs against ancestral virus (averagedifference of all dose groups). In comparison to vaccination withCV2CoV, VNTs induced by CV2CoV.351 were increased by a factor of 42 onday 21 (average difference of all dose groups). FIG. 6E shows that theB.1.351 variant vaccine CV2CoV.351 induced dose-dependent VNTs againstancestral SARS-CoV-2 (heterologous response) on day 42 in all dosegroups. Slightly higher responses except for all vaccinations with0.5 μg(group F) were shown upon vaccination with CV2CoV (homologous response).FIG. 6F shows that CV2CoV.351 induces dose-dependent VNTs againstB.1.351 SARS-CoV-2 (homologous response) on day 42 in all dose groups.In comparison to vaccination with CV2CoV, VNTs induced by CV2CoV.351were increased on day 42. FIG. 6G shows that the B.1.351 variant vaccineCV2CoV.351 induces dose-dependent VNTs against B.1.1.7 variantSARS-CoV-2 (heterologous response) on day 42 in all dose groups. Similarresponses for group H except for vaccination with 0.5 μg (group F) wereshown upon vaccination with CV2CoV (homologous response). FIG. 6H showsthat CV2CoV.351 induces dose-dependent VNTs against B.1.1.28 P.1 variantSARS-CoV-2 (homologous response) on day 42 in all dose groups. Lowerresponses were seen upon vaccination with CV2CoV (homologous response).

FIG. 7 shows significant IgG1 and IgG2a binding antibody responses onday 14 (FIG. 7A-D) for the groups vaccinated with bivalent mRNA vaccinecomposition CV2CoV+CV2CoV.351 formulated in LNPs. On day 14, FIG. 7A(IgG1 titer) and 7B (IgG2a titer) show dose-dependent levels of bindingantibody titers using doses of 0.5 μg, 2 μg and 8 μg (SARS-CoV-2ancestral receptor binding domain (RBD) protein coating). On day 14,FIG. 7C (IgG1 titer) and 7D (IgG2a titer) show dose-dependent levels ofbinding antibody titers using doses of 0.5 μg, 2 μg and 8 μg (B.1.351RBD variant K417N, E484K, N501Y protein coating). Significant inductionof VNTs assessed in a CPE-based assay is shown using ancestralSARS-CoV-2 (FIGS. 7E, 7F and 7I) or B.1.351 variant SARS-CoV-2 (FIGS.7G, 7H and 7J) on day 14, day 21 and day 42 respectively. FIGS. 7K and7L also show significant induction of VNTs assessed in a CPE-based assayusing 1.1.7 variant SARS-CoV-2 (FIG. 7K) or B.1.1.28 P.1 variant (FIG.7L) on day 42.

FIG. 8 shows of VNTs assessed in a CPE-based assay using ancestralSARS-CoV-2 (FIG. 8A) or using B.1.351 variant SARS-CoV-2 (FIG. 8B).Boost with CV2CoV or B.1.351 variant vaccine CV2CoV.351 shows strongboost capacity against ancestral SARS-CoV-2 and B.1.351 variantSARS-CoV-2 for homologous and heterologous response. Homologous responseis shown in FIG. 8A for group B, D and F and in FIG. 8B for group C, Eand G. Heterologous response is shown in FIG. 8A for group C, E and Gand in FIG. 8B for group B, D and F. Virus-neutralizing responsesagainst ancestral SARS-CoV-2 as well as against SARS-CoV-2 B.1.1.7(alpha), B.1.351 (beta) and P.1 (gamma) variants were tested 14 daysafter boosting (FIG. 8C-8F) on day 119 (VNTs against ancestralSARS-CoV-2 (FIG. 8C), against SARS-CoV-2 B.1.351 (FIG. 8D), SARS-CoV-2B.1.1.7 (FIG. 8E) and against P.1 (FIG. 8F).

FIG. 9 shows significant total IgG spike-binding antibody responses tothe ancestral SARS-CoV-2 RBD (FIG. 9A) and the B.1.351 RBD variantK417N, E484K, N501Y (FIG. 9B) on day 14 for all groups. Induction ofVNTs against different SARS-CoV-2 variants over time is shown in FIGS.9C-F (FIG. 9C: ancestral; FIG. 9D: B.1.1.7; FIG. 9E: B.1.351; FIG. F:P1). FIGS. 9G-J show cellular immune responses of CD8 (FIGS. 9G and 9I)and CD4 (FIGS. 9H and 9J) positive T-cells in mice stimulated with amixture of ancestral SARS-CoV-2 peptide library (FIGS. 9G and H) orstimulated with a mixture of B.1.351 SARS-CoV-2 peptide library (FIGS.9I and J), using an intracellular cytokine staining assay.

FIG. 10 shows cellular immune responses of CD8 (FIG. 10B) and CD4 (FIG.10A) positive T-cells in mice stimulated with a mixture of ancestralSARS-CoV-2 peptide library, using an intracellular cytokine stainingassay.

FIG. 11 shows VNTs upon prime and boost vaccination with CVnCoV orCV2CoV and a third Vaccination with bivalent CV2CoV+CV2CoV.351 vaccinecomposition in rats over time up to day 133 after first vaccination(FIG. 11A: VNTs against ancestral SARS-CoV-2, FIG. 11B: VNTs againstSARS-CoV-2 B.1.351). Robust and high VNTs on day 119 were induced notonly against ancestral and B.1.351 SARS-CoV-2, but also against B.1.1.7and P.1 SARS-CoV-2 variants (FIG. 11C: ancestral, FIG. 11D: B.1.351,FIG. 11E: B.1.1.7, FIG. 11F: P.1).

FIG. 12 shows antibody responses upon vaccination with vaccinecompositions encoding different mRNA formats of stabilized spike (S_stabpp) of delta variant SARS-CoV-2 B1.617.2 in rats (FIGS. 12A and 12B:Spike-binding antibodies detected via ELISA against delta B.1.617.2variant RBD on day 14 and day 42, respectively; FIGS. 12C, 12D, 12E:VNTs against SARS-CoV-2 B.1.617.2 on day 14, day 21, and day 42,respectively). Robust VNTs were induced not only against homologousSARS-CoV-2 variant (B.1.617.2) but also against heterologous SARS-CoV-2ancestral and SARS-CoV-2 variants B.1.351 and P.1 (FIG. 12F: ancestral,FIG. G: B.1.351, FIG. 12H: P.1).

FIG. 13 shows early antibody responses (total IgG) on day 14 uponvaccination with vaccine compositions encoding different mRNA constructencoding S_stab pp of different variant SARS-CoV-2 in rats. Furthermore,the bivalent approaches compare chemically modified mRNA withnon-modified mRNA (FIG. 13A: ancestral RBD; FIG. 13B: delta RBD (L452R,T478K); FIG. 13C: beta RBD (K417N, E484K, N501Y).

FIG. 14 shows vaccine efficacy by challenging mice with eitherSARS-CoV-2 variant B.1.351 or SARS CoV-2 variant B.1.627.2. Survival ofchallenged mice is shown in FIG. 14A: challenge with B.1.351 and FIG.14B: challenge with B.1.617.2. Mean percentage body weight changes areshown in FIG. 14C: challenge with B.1.351 and FIG. 14D: challenge withB.1.617.2. Viral RNA load in saliva is shown in FIG. 14E: B.1.351challenge group, and FIG. 14F: B.1.617.2 challenge group. Viral load inthe upper respiratory tract (URT) (conchae) demonstrates FIG. 14G forB.1.351 challenge group and FIG. 14H for B.617.2 challenge group) and inthe lower respiratory tract (LRT) (lung) in FIG. 14I (challenge withB.1.351) and J (challenge with B.1.617.2). Viral load in brain is shownin FIG. 14K to FIG. N (FIGS. 14K and L for cerebellum, FIGS. 14M and Nfor Cerebrum (for challenge group B.1.351: FIGS. 14K and M, forB.1.617.2: FIGS. 14L and N). Induction of anti-RBD total immunoglobulinsis shown in FIG. 14O: challenge group B.1.351 and FIG. 14P: challengegroup B.1.617.2. and VNTs in FIG. 14Q: post-challenge group B.1.351,FIG. 14R: pre-challenge group B.1.617.2, and FIG. 14S: post-challengegroup B.1.617.2.

FIG. 15 shows vaccine efficacy in with SARS-CoV-2 variant B.1.351challenged hamsters. FIG. 15A demonstrate the percentage body weightchanges in days post challenge infection. Viral RNA load in saliva isshown in FIG. 15B and in lung tissues in FIG. 15C. FIG. 15D demonstratesinduction of anti-RBD total immunoglobulins (Ig) and FIG. 15E VNTs.

DEFINITIONS

For the sake of clarity and readability, the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Percentages in the context of numbers should be understood as relativeto the total number of the respective items. In other cases, and unlessthe context dictates otherwise, percentages should be understood aspercentages by weight (wt.-%).

About: The term “about” is used when determinants or values do not needto be identical, i.e. 100% the same. Accordingly, “about” means, that adeterminant or values may diverge by 0.1% to 20%, or 0.1% to 10%,including any point within these ranges; e.g. by 0.1%, 0.2%, 0.3% 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. The skilled personwill know that e.g. certain parameters or determinants may slightly varybased on the method how the parameter was determined. For example, if acertain determinants or value is defined herein to have e.g. a length of“about 1000 nucleotides”, the length may diverge by 0.1% to 20%, or 0.1%to 10%, including any point within these ranges; e.g. by 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.Accordingly, the skilled person will know that in that specific examplesthe length may diverge by 1 to 200 nucleotides, or by 1 to 100nucleotides; in particular, by 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200nucleotides, or any integer value within the range.

Adaptive immune response: The term “adaptive immune response” as usedherein will be recognized and understood by the person of ordinary skillin the art, and is e.g. intended to refer to an antigen-specificresponse of the immune system (the adaptive immune system). Antigenspecificity allows for the generation of responses that are tailored tospecific pathogens or pathogen-infected cells. The ability to mountthese tailored responses is usually maintained in the body by “memorycells” (B-cells). In the context of the invention, the antigen isprovided by an RNA encoding at least one antigenic peptide or proteinderived from SARS-CoV-2, e.g. from a SARS-Cov-2 strain including, butnot limited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US),B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India),AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, SouthAfrica), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429(Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czechrepublic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1(Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1(Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu, Columbia).Preferably the antigen is provided by an RNA encoding at least oneantigenic peptide derived from a SARS-CoV-2 spike protein, e.g.comprising a spike protein from derived from a SARS-Cov-2 strainincluding, but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron,South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2,BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1(Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India),AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351(Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil),B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria),B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda),B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil),C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu,Columbia)

Antigen: The term “antigen” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a substance which may be recognized by the immunesystem, preferably by the adaptive immune system, and is capable oftriggering an antigen-specific immune response, e.g. by formation ofantibodies and/or antigen-specific T cells as part of an adaptive immuneresponse. Typically, an antigen may be or may comprise a peptide orprotein, which may be presented by the MHC to T-cells. Also fragments,variants and derivatives of peptides or proteins derived from e.g. spikeprotein (S) of SARS-CoV-2, e.g from a spike protein (S) of a SARS-Cov-2strain including, but not limited to: C.1.2 (South Africa), B.1.1.529(Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2,BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619(Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India),AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3(India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma,Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta,Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1(Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta,Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621(Mu, Columbia) comprising at least one epitope are understood asantigens in the context of the invention. In the context of the presentinvention, an antigen may be the product of translation of a providedRNA as specified herein.

Antigenic peptide or protein: The term “antigenic peptide or protein” or“immunogenic peptide or protein” will be recognized and understood bythe person of ordinary skill in the art, and is e.g. intended to referto a peptide, protein derived from a (antigenic or immunogenic) proteinwhich stimulates the body's adaptive immune system to provide anadaptive immune response. Therefore, an antigenic/immunogenic peptide orprotein comprises at least one epitope or antigen of the protein it isderived from (e.g., spike protein (S) of SARS-CoV-2), e.g. derived froma spike protein (S) from a SARS-Cov-2 strain including, but not limitedto: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa) (includingBA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5),C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176(Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India), AY.4.2 (DeltaPlus, India), B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7(Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California,US), B.1.525 (Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota,New York, US), A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2(Delta, India), P.2 (Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta,Philippines), and/or B.1.621 (Mu, Columbia).

Cationic: Unless a different meaning is clear from the specific context,the term “cationic” means that the respective structure bears a positivecharge, either permanently or not permanently, but in response tocertain conditions such as pH. Thus, the term “cationic” covers both“permanently cationic” and “cationisable”.

Cationisable: The term “cationisable” as used herein means that acompound, or group or atom, is positively charged at a lower pH anduncharged at a higher pH of its environment. Also in non-aqueousenvironments where no pH value can be determined, a cationisablecompound, group or atom is positively charged at a high hydrogen ionconcentration and uncharged at a low concentration or activity ofhydrogen ions. It depends on the individual properties of thecationisable or polycationisable compound, in particular the pKa of therespective cationisable group or atom, at which pH or hydrogen ionconcentration it is charged or uncharged. In diluted aqueousenvironments, the fraction of cationisable compounds, groups or atomsbearing a positive charge may be estimated using the so-calledHenderson-Hasselbalch equation, which is well-known to a person skilledin the art. E.g., in some embodiments, if a compound or moiety iscationisable, it is preferred that it is positively charged at a pHvalue of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, morepreferably of a pH value of or below 9, of or below 8, of or below 7,most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e.under physiological conditions, particularly under physiological saltconditions of the cell in vivo. In other embodiments, it is preferredthat the cationisable compound or moiety is predominantly neutral atphysiological pH values, e.g. about 7.0-7.4, but becomes positivelycharged at lower pH values. In some embodiments, the preferred range ofpKa for the cationisable compound or moiety is about 5 to about 7.

Coding sequence/coding region: The terms “coding sequence” or “codingregion” and the corresponding abbreviation “cds” as used herein will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a sequence of several nucleotidetriplets, which may be translated into a peptide or protein. A codingsequence in the context of the present invention may be an RNA sequenceconsisting of a number of nucleotides that may be divided by three,which starts with a start codon and which preferably terminates with astop codon.

Derived from: The term “derived from” as used throughout the presentspecification in the context of a nucleic acid, i.e. for a nucleic acid“derived from” (another) nucleic acid, means that the nucleic acid,which is derived from (another) nucleic acid, shares e.g. at least 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleicacid from which it is derived. The skilled person is aware that sequenceidentity is typically calculated for the same types of nucleic acids,i.e. for DNA sequences or for RNA sequences. Thus, it is understood, ifa DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, ina first step the RNA sequence is converted into the corresponding DNAsequence (in particular by replacing the uracils (U) by thymidines (T)throughout the sequence) or, vice versa, the DNA sequence is convertedinto the corresponding RNA sequence (in particular by replacing the T byU throughout the sequence). Thereafter, the sequence identity of the DNAsequences or the sequence identity of the RNA sequences is determined.Preferably, a nucleic acid “derived from” a nucleic acid also refers tonucleic acid, which is modified in comparison to the nucleic acid fromwhich it is derived, e.g. in order to increase RNA stability evenfurther and/or to prolong and/or increase protein production. In thecontext of amino acid sequences (e.g. antigenic peptides or proteins)the term “derived from” means that the amino acid sequence, which isderived from (another) amino acid sequence, shares e.g. at least 60%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with theamino acid sequence from which it is derived.

Epitope: The term “epitope” (also called “antigen determinant” in theart) as used herein will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to refer to T cellepitopes and B cell epitopes. T cell epitopes or parts of the antigenicpeptides or proteins and may comprise fragments preferably having alength of about 6 to about 20 or even more amino acids, e.g. fragmentsas processed and presented by MHC class I molecules, preferably having alength of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even11, or 12 amino acids), or fragments as processed and presented by MHCclass II molecules, preferably having a length of about 13 to about 20or even more amino acids. These fragments are typically recognized by Tcells in form of a complex consisting of the peptide fragment and an MHCmolecule, i.e. the fragments are typically not recognized in theirnative form. B cell epitopes are typically fragments located on theouter surface of (native) protein or peptide antigens, preferably having5 to 15 amino acids, more preferably having 5 to 12 amino acids, evenmore preferably having 6 to 9 amino acids, which may be recognized byantibodies, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context epitopes can beconformational or discontinuous epitopes which are composed of segmentsof the proteins or peptides as defined herein that are discontinuous inthe amino acid sequence of the proteins or peptides as defined hereinbut are brought together in the three-dimensional structure orcontinuous or linear epitopes which are composed of a single polypeptidechain.

Fragment: The term “fragment” as used throughout the presentspecification in the context of a nucleic acid sequence (e.g. RNA or aDNA) or an amino acid sequence may typically be a shorter portion of afull-length sequence of e.g. a nucleic acid sequence or an amino acidsequence, while still retaining its intended function. Accordingly, afragment, typically, consists of a sequence that is identical to thecorresponding stretch within the full-length sequence. A preferredfragment of a sequence in the context of the present invention, consistsof a continuous stretch of entities, such as nucleotides or amino acidscorresponding to a continuous stretch of entities in the molecule thefragment is derived from, which represents at least 40%, 50%, 60%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% of the total (i.e. full-length)molecule from which the fragment is derived (e.g. spike protein (S) ofSARS-CoV-2, e.g. from spike protein (S) of a SARS-Cov-2 strainincluding, but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron,South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2,BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1(Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India),AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351(Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil),B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria),B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda),B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil),C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu,Columbia)). The term “fragment” as used throughout the presentspecification in the context of proteins or peptides may, typically,comprise a sequence of a protein or peptide as defined herein, which is,with regard to its amino acid sequence, N-terminally and/or C-terminallytruncated compared to the amino acid sequence of the original protein.Such truncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide.Fragments of proteins or peptides may comprise at least one epitope ofthose proteins or peptides.

Heterologous: The terms “heterologous” or “heterologous sequence” asused throughout the present specification in the context of a nucleicacid sequence or an amino acid sequence refers to a sequence (e.g. RNA,DNA, amino acid) has to be understood as a sequence that is derived fromanother gene, another allele, or e.g. another species or virus. Twosequences are typically understood to be “heterologous” if they are notderivable from the same gene or from the same allele. I.e., althoughheterologous sequences may be derivable from the same organism or virus,in nature, they do not occur in the same nucleic acid or protein.

Humoral immune response: The terms “humoral immunity” or “humoral immuneresponse” will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to refer to B-cell mediatedantibody production and optionally to accessory processes accompanyingantibody production. A humoral immune response may be typicallycharacterized, e.g. by Th2 activation and cytokine production, germinalcenter formation and isotype switching, affinity maturation and memorycell generation. Humoral immunity may also refer to the effectorfunctions of antibodies, which include pathogen and toxinneutralization, classical complement activation, and opsonin promotionof phagocytosis and pathogen elimination.

Identity (of a sequence): The term “identity” as used throughout thepresent specification in the context of a nucleic acid sequence or anamino acid sequence will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to refer to thepercentage to which two sequences are identical over the full/entirelength thereof or over a specific designated portion, region or domainthereof. For example, there is at least 40%, 50%, 60%, 70%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5% identity over the full/entire length thereofor over a specific designated portion, region or domain thereof. Todetermine the percentage to which two sequences are identical, e.g.nucleic acid sequences or amino acid (aa) sequences as defined herein,preferably the aa sequences encoded by the nucleic acid sequence asdefined herein or the aa sequences themselves, the sequences can bealigned in order to be subsequently compared to one another. Therefore,e.g. a position of a first sequence may be compared with thecorresponding position of the second sequence. If a position in thefirst sequence is occupied by the same residue as is the case at aposition in the second sequence, the two sequences are identical at thisposition. If this is not the case, the sequences differ at thisposition. If insertions occur in the second sequence in comparison tothe first sequence, gaps can be inserted into the first sequence toallow a further alignment. If deletions occur in the second sequence incomparison to the first sequence, gaps can be inserted into the secondsequence to allow a further alignment. The percentage to which twosequences are identical is then a function of the number of identicalpositions divided by the total number of positions including thosepositions which are only occupied in one sequence. The percentage towhich two sequences are identical can be determined using an algorithm,e.g. an algorithm integrated in the BLAST program.

Immunogen, immunogenic: The terms “immunogen” or “immunogenic” will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a compound that is able tostimulate/induce an immune response. Preferably, an immunogen may be apeptide, polypeptide, or protein. An immunogen in the sense of thepresent invention is the product of translation of a provided RNAcomprising at least one coding sequence encoding at least one antigenicpeptide, protein derived from spike protein of SARS-CoV-2, e.g. aprotein derived from a spike protein of of a SARS-Cov-2 strainincluding, but not limited to: C.1.2 (South Africa), B.1.1.529 (Omicron,South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2,BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1(Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India),AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351(Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil),B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta, Nigeria),B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1 (Uganda),B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil),C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu,Columbia) as defined herein. Typically, an immunogen elicits an adaptiveimmune response.

Immune response: The term “immune response” will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a specific reaction of the adaptive immune systemto a particular antigen (so called specific or adaptive immune response)or an unspecific reaction of the innate immune system (so calledunspecific or innate immune response), or a combination thereof. Asuitable vaccine induces an efficient immune response in a normalhealthy recipient to whom the vaccine is administered. With an efficientimmune response one vaccination will result in virus-neutralizingantibody titers. In addition, or alternatively, an efficient immuneresponse will elicit an adaptive immune response. In some embodimentsthe efficient immune response will reduce coronavirus infection by atleast 50% relative to a neutralizing antibody titer of an unvaccinatedcontrol subject. In some embodiments, an efficient immune response willbe one where the neutralizing antibody titer and/or a T cell immuneresponse is sufficient to reduce the rate of asymptomatic viralinfection relative to the neutralizing antibody titer of unvaccinatedcontrol subjects. An efficient immune response may also be one where theneutralizing antibody titer and/or a T cell immune response issufficient to prevent viral latency in the subject and/or theneutralizing antibody titer is sufficient to block fusion of virus withepithelial cells of the subject. In some embodiments an efficient immuneresponse is one in which administration of a therapeutically effectiveamount of the nucleic acid, the composition, the polypeptide, or thevaccine to a subject induces a T cell immune response againstcoronavirus in the subject. In preferred embodiments, the T cell immuneresponse comprises a CD4+ T cell immune response and/or a CD8+ T cellimmune response. In further aspects, an efficient immune response is onein which the immune response protects the subject from severe COVID-19disease for at least about 6 months and/or reduce the incidence ofhospitalization compared to an unvaccinated person. An efficient immuneresponse may also reduce the transmission of virus due compared totransmission from an unvaccinated person infected with the virus. Anefficient immune response may also be considered as one which providesome protection against variants due to heterologous immune responses.

Immune system: The term “immune system” will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a system of the organism that may protect theorganisms from infection. If a pathogen succeeds in passing a physicalbarrier of an organism and enters this organism, the innate immunesystem provides an immediate, but non-specific response. If pathogensevade this innate response, vertebrates possess a second layer ofprotection, the adaptive immune system. Here, the immune system adaptsits response during an infection to improve its recognition of thepathogen. This improved response is then retained after the pathogen hasbeen eliminated, in the form of an immunological memory, and allows theadaptive immune system to mount faster and stronger attacks each timethis pathogen is encountered. According to this, the immune systemcomprises the innate and the adaptive immune system. Each of these twoparts typically contains so called humoral and cellular components.

Innate immune system: The term “innate immune system” (also known asnon-specific or unspecific immune system) will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a system typically comprising the cells andmechanisms that defend the host from infection by other organisms in anon-specific manner. This means that the cells of the innate system mayrecognize and respond to pathogens in a generic way, but unlike theadaptive immune system, it does not confer long-lasting or protectiveimmunity to the host. The innate immune system may be activated byligands of pattern recognition receptor e.g. Toll-like receptors,NOD-like receptors, or RIG-I like receptors etc.

Lipidoid compound: A lipidoid compound, also referred to as lipidoid, isa lipid-like compound, i.e. an amphiphilic compound with lipid-likephysical properties. In the context of the present invention, the termlipid is considered to encompass lipidoid compounds.

Permanently cationic: The term “permanently cationic” as used hereinwill be recognized and understood by the person of ordinary skill in theart, and means, e.g., that the respective compound, or group, or atom,is positively charged at any pH value or hydrogen ion activity of itsenvironment. Typically, the positive charge results from the presence ofa quaternary nitrogen atom. Where a compound carries a plurality of suchpositive charges, it may be referred to as permanently polycationic.

RNA sequence: The term “RNA sequence” will be recognized and understoodby the person of ordinary skill in the art, and e.g. refer to aparticular and individual order of the succession of itsribonucleotides.

Stabilized RNA: The term “stabilized RNA” refers to an RNA that ismodified such that it is more stable to disintegration or degradation,e.g., by environmental factors or enzymatic digest, such as by exo- orendonuclease degradation, compared to an RNA without such modification.Preferably, a stabilized RNA in the context of the present invention isstabilized in a cell, such as a prokaryotic or eukaryotic cell,preferably in a mammalian cell, such as a human cell. The stabilizationeffect may also be exerted outside of cells, e.g. in a buffer solutionetc., e.g., for storage of a composition comprising the stabilized RNA.

T-cell responses: The terms “cellular immunity” or “cellular immuneresponse” or “cellular T-cell responses” as used herein will berecognized and understood by the person of ordinary skill in the art,and are for example intended to refer to the activation of macrophages,natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, andthe release of various cytokines in response to an antigen. In moregeneral terms, cellular immunity is not based on antibodies, but on theactivation of cells of the immune system. Typically, a cellular immuneresponse may be characterized e.g. by activating antigen-specificcytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g.specific immune cells like dendritic cells or other cells, displayingepitopes of foreign antigens on their surface.

UTR: The term “untranslated region” or “UTR” or “UTR element” will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a part of a nucleic acid moleculetypically located 5′ or 3′ located of a coding sequence. An UTR is nottranslated into protein. An UTR may be part of a nucleic acid, e.g. aDNA or an RNA. An UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, e.g., ribosomal binding sites, miRNA binding sites etc.

3′-UTR: The term “3′-untranslated region” or “3′-UTR” or “3′-UTRelement” will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to refer to a part of a nucleicacid molecule located 3′ (i.e. downstream) of a coding sequence andwhich is not translated into protein. A 3′-UTR may be part of an RNA,located between a coding sequence and an (optional) poly(A) sequence. A3′-UTR may comprise elements for controlling gene expression, alsocalled regulatory elements. Such regulatory elements may be, e.g.,ribosomal binding sites, miRNA binding sites etc.

5′-UTR: The term “5′-untranslated region” or “5′-UTR” or “5′-UTRelement” will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to refer to a part of a nucleicacid molecule located 5′ (i.e. upstream) of a coding sequence and whichis not translated into protein. A 5′-UTR may be part of an RNA, locatedbetween a coding sequence and an (optional) 5′ cap. A 5′-UTR maycomprise elements for controlling gene expression, also calledregulatory elements. Such regulatory elements may be, e.g., ribosomalbinding sites, miRNA binding sites etc.

Variant (of a sequence): The term “variant” as used throughout thepresent specification in the context of a nucleic acid sequence will berecognized and understood by the person of ordinary skill in the art,and is e.g. intended to refer to a variant of a nucleic acid sequencederived from another nucleic acid sequence. E.g., a variant of a nucleicacid sequence may exhibit one or more nucleotide deletions, insertions,additions and/or substitutions compared to the nucleic acid sequencefrom which the variant is derived. A variant of a nucleic acid sequencemay at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%identical to the nucleic acid sequence the variant is derived from. Thevariant is a functional variant in the sense that the variant hasretained at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%or more of the function of the sequence where it is derived from. In oneembodiment a “variant” of a nucleic acid sequence may have at least 40%,50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% nucleotide identityover a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotide of suchnucleic acid sequence.

The term “variant” as used throughout the present specification in thecontext of proteins or peptides is e.g. intended to refer to a proteinsor peptide variant having an amino acid sequence which differs from theoriginal sequence in one or more mutation(s)/substitution(s), such asone or more substituted, inserted and/or deleted amino acid(s). Forexample, in some aspects an insertion in a protein sequence comprises aninsertion of 1 to 10 amino acids, such 1, 2, 3, 4, 5, 6,7 8, 9 or 10consecutive amino acids. Preferably, these fragments and/or variants mayhave the same, or a comparable specific antigenic property (immunogenicvariants, antigenic variants). Insertions and substitutions arepossible, in particular, at those sequence positions which cause nomodification to the three-dimensional structure or do not affect thebinding region. Modifications to a three-dimensional structure byinsertion(s) or deletion(s) can easily be determined e.g. using CDspectra (circular dichroism spectra). A “variant” of a protein orpeptide may have at least 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5% amino acid identity over a stretch of at least 10, 20, 30,50, 75 or 100 amino acids or over the entire length of such protein orpeptide. Preferably, a variant of a protein may comprise a functionalvariant of the protein, which means, in the context of the invention,that the variant exerts essentially the same, or at least 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the immunogenicity as theprotein it is derived from.

SHORT DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the finding that RNAencoding spike proteins derived from SARS-CoV-2 variants can beefficiently expressed in human cells and induce an antibody response inanimals that broadly neutralizes different SARS-CoV-2 variants, e.g. aSARS-Cov-2 strain including, but not limited to: C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia). Moreover, mixtures of RNA encoding differentSARS-CoV-2 spike protein variants are also shown to be effective inproducing neutralizing antibodies to a range of SARS-CoV-2 variants.These findings provide basis for new RNA-based coronavirus vaccines.

RNA sequences, composition, or vaccines as described herein have atleast some of the following advantageous features:

-   -   Translation of the RNA at the site of injection/vaccination        (e.g. muscle);    -   Very efficient induction of antigen-specific immune responses        against the encoded SARS-CoV-2 protein at a very low dosage and        dosing regimen;    -   Suitability for vaccination of infants and/or newborns or the        elderly, in particular the elderly;    -   Suitability of the composition/vaccine for intramuscular        administration;    -   Induction of specific and functional humoral immune response        against SARS-CoV-2 variants;    -   Induction of broad, functional cellular T-cell responses against        against SARS-CoV-2 variants;    -   Induction of specific B-cell memory against SARS-CoV-2 variants;    -   Induction of functional antibodies that can effectively        neutralize the SARS-CoV-2 virus variants;    -   Induction of functional antibodies that can also effectively        neutralize the original SARS-CoV-2 virus;    -   Eliciting of mucosal IgA immunity by inducing of mucosal IgA        antibodies;    -   Induction of a well-balanced B cell and T cell responses;    -   Induction of protective immunity against SARS-CoV-2 variants;    -   Fast onset of immune protection against SARS-CoV-2 variants;    -   Longevity of the induced immune responses against SARS-CoV-2        variants;    -   No enhancement of a SARS-CoV-2 infection due to vaccination or        immunopathological effects;    -   No antibody dependent enhancement (ADE) caused by the RNA based        SARS-CoV-2 vaccine;    -   No excessive induction of systemic cytokine or chemokine        response after application of the vaccine, which could lead to        an undesired high reactogenicity upon vaccination;    -   Well tolerability, no side-effects, non-toxicity of the vaccine;    -   Advantageous stability characteristics of the RNA-based vaccine;    -   Speed, adaptability, simplicity and scalability of SARS-CoV-2        variant vaccine production;    -   Advantageous vaccination regimen that only requires one or two        vaccination for sufficient protection;    -   Advantageous vaccination regimen that only requires a low dose        of the vaccine for sufficient protection;    -   Advantageous vaccination regimen that only requires a low dose        of the composition/vaccine for sufficient protection which        allows the combination of different antigen providing RNAs for        multivalent vaccines;    -   Boostability of an existing immunity against SARS-CoV-2,        preferably inducing additional immune responses against        SARS-CoV-2 variants;    -   Induction of different, SARS-CoV-2 strain specific immune        responses in subjects that have been exposed to a different a        strain or that have been vaccinated with a vaccine against a        different strain;    -   Induction of a broad immune response across various SARS-CoV-2        variants;

In a first aspect, the present invention provides an RNA encoding atleast one SARS-CoV-2 spike protein or an immunogenic fragment orimmunogenic variant thereof, wherein the SARS-CoV-2 spike proteincomprises at least one amino acid substitution, deletion or insertion ata position corresponding to H69; V70; A222; Y453; S477; I692; R403;K417; N437; N439; V445; G446; L455; F456; K458; A475; G476; T478; E484;G485, F486; N487; Y489; F490; Q493; S494; P499; T500; N501; V503; G504;Y505; Q506; Y144; A570; P681; T716; S982; D1118; L18; D80; D215; L242;A243; L244; R246; A701; T20; P26; D138; R190; H655; T1027; S13; W152;L452; R346; P384; G447; G502; T748; A522; V1176; T859; S247; Y248; L249;T250; P251; G252; G75; T76; D950; E154; G769; S254; Q613; F157; R158;Q957; D253; T95; F888; Q677; A67; Q414; N450; V483; G669; T732; Q949;Q1071; E1092; H1101; N1187; W258; T19; V126; H245; S12; A899; G142;E156; K558; and/or Q52 relative to the sequence of SEQ ID NO: 1, whereinthe RNA comprises at least one heterologous untranslated region. Incertain embodiments, the RNA encodes a SARS-CoV-2 spike protein thatcomprises at least one amino acid substitution, deletion or insertion ata position from a SARS-CoV-2 variant spike protein (e.g. from aSARS-Cov-2 strain including, but not limited to: C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia).

In a second aspect, the present invention provides a composition,preferably an immunogenic composition comprising at least one RNA of thefirst aspect. Suitably, the composition comprises at least one RNA ofthe first aspect formulated in lipid-based carriers, preferably in lipidnanoparticles (LNPs). In preferred embodiments, the second aspectrelates to multivalent compositions, such as compositions comprisingRNAs encoding SARS-CoV-2 spike proteins having different amino acidcoding sequences (e.g., spike proteins from more than one SARS-CoV-2strain, including more than one SARS-CoV-2 variant strain, e.g. spikeproteins from more than one more SARS-Cov-2 strain including, but notlimited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US),B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India),AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, SouthAfrica), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429(Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czechrepublic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1(Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1(Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu,Columbia)).

In a third aspect, the present invention provides a SARS-CoV-2 variantvaccine, wherein the vaccine comprises at least one RNA of the firstaspect, or at least one composition of the second aspect. In preferredembodiments, the second aspect relates to multivalent SARS-CoV-2vaccines. In preferred embodiments, the third aspect relates toSARS-CoV-2 variant booster vaccines. The SARS-CoV-2 variant boostervaccines may be for one or more SARS-Cov-2 strains including, but notlimited to: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US),B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India),AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, SouthAfrica), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429(Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czechrepublic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1(Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1(Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu, Columbia)

In a fourth aspect, the present invention provides a kit or kit of partscomprising at least one RNA of the first aspect, and/or at least onecomposition of the second aspect, and/or at least one SARS-CoV-2 variantvaccine of the third aspect.

In a fifth aspect, the present invention provides a combinationcomprising at least two separate components, wherein the at least twoseparate components are each RNA species of the first aspect, and/orcompositions of the second aspect, and/or SARS-CoV-2 variant vaccines ofthe third aspect, i.e. each component is a RNA species, compositionand/or SARS-Cov-2 variant vaccine directed to a different SARS-Cov-2,wherein said two separate components may be each directed to aSARS-Cov-2 variant including, but not limited to: C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia).

Further aspects of the invention concern a method of treating orpreventing a SARS-CoV-2 infection in a subject, and first and secondmedical uses of nucleic acid, compositions, and vaccines. Also providedare methods of manufacturing the nucleic acid, the composition, or thevaccine.

DETAILED DESCRIPTION OF THE INVENTION

The present application is filed together with a sequence listing inelectronic format, which is part of the description of the presentapplication (WIPO standard ST.25). The information contained in thesequence listing is incorporated herein by reference in its entirety.Where reference is made herein to a “SEQ ID NO”, the correspondingnucleic acid sequence or amino acid (aa) sequence in the sequencelisting having the respective identifier is referred to. For manysequences, the sequence listing also provides additional detailedinformation, e.g. regarding certain structural features, sequenceoptimizations, GenBank (NCBI) or GISAID (epi) identifiers, or additionaldetailed information regarding its coding capacity. In particular, suchinformation is provided under numeric identifier <223> in the WIPOstandard ST.25 sequence listing. Accordingly, information provided undersaid numeric identifier <223> is explicitly included herein in itsentirety and has to be understood as integral part of the description ofthe underlying invention.

RNA Suitable for a SARS-CoV-2 Variant Vaccine:

In a first aspect, the invention relates to an RNA suitable for aSARS-CoV-2 variant vaccine.

It has to be noted that specific features and embodiments that aredescribed in the context of the first aspect of the invention, that isthe RNA of the invention, are likewise applicable to the second aspect(composition of the invention), the third aspect (vaccine of theinvention), the fourth aspect (kit or kit of parts of the invention),the fifth aspect (combination of the invention), or further aspectsincluding medical uses and method of treatments.

The RNA of the first aspect forms the basis for an RNA based compositionor vaccine. Generally, protein-based vaccines, or live attenuatedvaccines, are suboptimal for use in developing countries due to theirhigh production costs. In addition, protein-based vaccines, or liveattenuated vaccines require long development times and are not suitablefor rapid responses of pandemic virus outbreaks such as the SARS-CoV-2outbreak in 2019/2020. In contrast, RNA-based vaccines according to thepresent invention allow very fast and cost-effective production.Therefore, in comparison with known vaccines, vaccine based on theinventive RNA can be produced significantly cheaper and faster, which isvery advantageous particularly for use in developing countries. Onefurther advantage of a vaccine based on RNA may be itstemperature-stability in comparison to protein or peptide-basedvaccines.

In particularly preferred embodiments, the first aspect of the inventionrelates to an RNA comprising at least one coding sequence encoding atleast one antigenic peptide or protein from a SARS-CoV-2 spike proteinor an immunogenic fragment or immunogenic variant thereof, wherein theRNA comprises at least one heterologous untranslated region (UTR) andwherein the SARS-CoV-2 spike protein comprises at least one amino acidsubstitution selected from a SARS-CoV-2 variant and optionally astabilizing mutation of a SARS-Co-2 strain.

The term “antigenic peptide or protein from a SARS-CoV-2 spike protein”herein means (i) an antigen that is a SARS-CoV-2 spike protein havingamino acid sequence of the antigenic peptide or protein (or a fragmentthereof) which is identical to a SARS-CoV-2 variant protein (or afragment thereof), or (ii) an antigen that is derived from a SARS-CoV-2spike protein having an amino acid sequence of the antigenic peptide orprotein (or a fragment thereof) which is not identical to acorresponding SARS-CoV-2 variant protein (or a fragment thereof). Forexample, the respective SARS-CoV-2 spike protein may comprise at leastone amino acid substitution, insertion or deletion selected from aSARS-CoV-2 variant and/or at least one pre-fusion stabilizing mutation.

The term “immunogenic fragment” or “immunogenic variant” herein meansany fragment/variant of the corresponding SARS-CoV-2 antigen that iscapable of raising an immune response in a subject. Preferably,intramuscular or intradermal administration of the RNA of the firstaspect results in expression of the encoded SARS-CoV-2 spike protein ina subject.

The term “expression” as used herein refers to the production of aSARS-CoV-2 spike protein, wherein said SARS-CoV-2 spike protein isprovided by a coding sequence of an RNA of the first aspect. Forexample, “expression” of an RNA refers to production of a protein (e.g.after administration of said RNA to a cell or a subject) via translationof the RNA into a polypeptide, e.g. into a peptide or protein that is oris derived from a SARS-CoV-2 coronavirus. The term “expression” and theterm “production” may be used interchangeably herein. Further, the term“expression” preferably relates to production of a certain peptide orprotein upon administration of an RNA to a cell or an organism.

In preferred embodiments, the RNA of the invention is suitable for aSARS-CoV-2 variant vaccine.

A SARS-CoV-2 Spike protein is a type I viral fusion protein that existsas trimer on the viral surface with each monomer consisting of a Head(S1) and stem (S2). Individual precursor S polypeptides form ahomotrimer and undergo glycosylation within the Golgi apparatus as wellas processing to remove the signal peptide, and cleavage by a cellularprotease to generate separate S1 and S2 polypeptide chains, which remainassociated as S1/S2 protomers within the homotrimer and is therefore atrimer of heterodimers. The S1 domain of the spike glycoprotein includesthe receptor binding domain (RBD) that engages (most likely) with theangiotensin-converting enzyme 2 receptors and mediates viral fusion intothe host cell, an N-terminal domain that may make initial contact withtarget cells, and 2 subdomains, all of which are susceptible toneutralizing antibodies. S2 domain consists of a six helix bundle fusioncore involved in membrane fusion with the host endosomal membrane and isalso a target for neutralization. The S2 subunit further comprises twoheptad-repeat sequences (HR1 and HR2) and a central helix typical offusion glycoproteins, a transmembrane domain, and the cytosolic taildomain.

In the context of the invention, any Spike protein that is selected fromor is derived from a SARS-CoV-2 variant and comprises least one aminoacid substitution, deletion or insertion when compared to SEQ ID NO:1may be used and may be suitably encoded by the coding sequence or theRNA of the first aspect. It is further in the scope of the underlyinginvention, that the at least one antigenic peptide or protein maycomprise or consist of a synthetically engineered or an artificialSARS-CoV-2 spike protein. The term “synthetically engineered” SARS-CoV-2spike protein, or the term “artificial SARS-CoV-2 spike protein” or theterm “recombinant” SARS-CoV-2 spike protein relates to a protein thatdoes not occur in nature. Accordingly, an “artificial SARS-CoV-2 spikeprotein” or a “synthetically engineered SARS-CoV-2 spike protein” or theterm “recombinant” SARS-CoV-2 spike protein may, for example, differ inat least one amino acid compared to a naturally occurring SARS-CoV-2spike protein (e.g., comprising one or more heterologous/introducedamino acids as compared to a naturally occurring SARS-CoV-2 spikeprotein), and/or may comprise an additional heterologous peptide orprotein element, and/or may be N-terminally or C-terminally extended ortruncated.

In the following, preferred antigenic peptide or protein sequences thatare provided by the RNA of the invention are described in detail.

It should be noted that where reference is made to amino acid (aa)residues and their position in a SARS-CoV-2 spike protein (S), anynumbering used herein—unless stated otherwise—relates to the position ofthe respective amino acid residue in a corresponding spike protein (S)of the original SARS-CoV-2 coronavirus isolate EPI_ISL_402128 accordingto SEQ ID NO: 1. Respective amino acid positions are, throughout thedisclosure, exemplarily indicated for spike protein (S) of the originalSARS-CoV-2 coronavirus isolate EPI_ISL_402128 (SEQ ID NO: 1).

Protein annotation as used herein relates to SEQ ID NO: 1 as a referenceprotein. The full-length spike protein (S) of the original SARS-CoV-2coronavirus reference protein has 1273 amino acid residues, andcomprises the following elements:

-   -   secretory signal peptide: amino acid position aa 1 to aa 15 (see        SEQ ID NO: 28)    -   spike protein fragment S1: amino acid position aa 1 to aa 681        (see SEQ ID NO: 27)    -   S1-N-Terminal Domain (S1-NTD) amino acid position aa 13 to aa        303 (see SEQ ID NO: 26992)    -   receptor binding domain (RBD): amino acid position aa 319 to aa        541 (see SEQ ID NO: 13243)    -   critical neutralisation domain (CND): amino acid position aa 329        to aa 529 (see SEQ ID NO: 13310)    -   spike protein fragment S2: amino acid position aa 682 to aa 1273        (see SEQ ID NO: 30)    -   transmembrane domain (TM) amino acid position aa 1212 to aa 1273        (see SEQ ID NO: 49)    -   transmembrane domain (TMflex) amino acid position aa 1148 to aa        1273 (see SEQ ID NO: 13176)    -   Furine cleavage site region (S1/S2) amino acid position aa 681        to aa 685 (see SEQ ID NO: 26994)

It should be noted that variation on an amino acid level naturallyoccurs between spike proteins derived from different SARS-CoV-2 isolatesor SARS-CoV-2 variants. In the context of the invention, such amino acidvariations can be applied to antigenic peptide or protein derived from aspike protein as described herein. Suitably, the amino acid variationsor mutations are selected in a way to 1) induce an immune responseagainst the SARS-CoV-2 virus variant the substitution/mutation isderived from and/or (2) to produce an antigenic peptide or protein thatis desirable for inducing an immune response (e.g., an antigenic peptideor protein derived from a spike protein and that is in a pre-fusionform).

Accordingly, in particularly preferred embodiments, the RNA of theinvention comprises at least one coding sequence encoding at least oneSARS-CoV-2 spike protein, or an immunogenic fragment or immunogenicvariant thereof, wherein the SARS-CoV-2 spike protein comprises at leastone amino acid substitution, deletion, or insertion selected from aSARS-CoV-2 variant.

In that context, the term “at least one amino acid substitution,deletion, or insertion selected from a SARS-CoV-2 variant” herein meansat least one amino acid position in the SARS-CoV-2 spike protein (orfragment thereof) that is different to the original SARS-CoV-2 spikeprotein (according to the SEQ ID NO: 1 reference strain).

In preferred embodiments, the SARS-CoV-2 variant is selected from or isderived from the following SARS-CoV-2 lineages: C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia).

In particularly preferred embodiments, the SARS-CoV-2 variant isselected from or derived from the following SARS-CoV-2 lineages: B.1.351(South Africa), P.1 (Brazil), B.1.617.1 (India), B.1.617.2 (India),B.1.617.3 (India), B.1.1.529 (Omicron, South Africa) (including BA.1_v1,BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).

Accordingly, each spike protein provided herein and contemplated assuitable antigen in the context of the invention may have one or more ofthe following amino acid variations or mutations (amino acid positionsaccording to reference SEQ ID NO: 1) as provided in List 1. Thevariations or mutations provided below are derived from new emergingSARS-CoV-2 virus variants, and may be integrated into the spike proteinthat is encoded by the RNA of the invention:

List 1A: Amino Acid Positions for Substitutions Deletions and/orInsertions

H69; V70; A222; Y453; S477; I692; R403; K417; N437; N439; V445; G446;L455; F456; K458; A475; G476; T478; E484; G485, F486; N487; Y489; F490;Q493; S494; P499; T500; N501; V503; G504; Y505; Q506; Y144; A570; P681;T716; S982; D1118; L18; D80; D215; L242; A243; L244; R246; A701; T20;P26; D138; R190; H655; T1027; S13; W152; L452; R346; P384; G447; G502;T748; A522; V1176; T859; S247; Y248; L249; T250; P251; G252; G75; T76;D950; E154; G769; S254; Q613; F157; R158; Q957; D253; T95; F888; Q677;A67; Q414; N450; V483; G669; T732; Q949; Q1071; E1092; H1101; N1187;W258; T19; V126; H245; S12; A899; G142; E156; K558; G339; P9; C136;Y449; L24; P25; P26; A27; V213; S371; T376; D405; A701; 1210; D936;S939; R357; R682; R683; A684; R685; V143, Y144, Y145, N211, L212, R214,E241, G339, S371, S373, S375, N440, G496, Q498, Y505, T547, D614, N679,P681, N764, D796, N856, Q954, N969, L981 or Q52 (relative to thesequence of SEQ ID NO: 1).

List 1B: Amino Acid Substitutions Deletions or Insertions

H69del; V70del; A222V; Y453F; S477N; I692V; R403K; K417N; N437S; N439K;V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V;G476S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T478R; T478A;E484Q; E484K; E484A; E484D; G485R; G485S, F486L; N487I; Y489H; F490S;F490L; Q493L; Q493K; S494P; S494L; P499L; T500I; N501Y; N501T; N501S;V503F; V503I; G504D; Y505W; Q506K; Q506H; Y144del; A570D; P681H; T716I;S982A; D1118H; L18F; D80A; D215G; L242del; A243del; L244del; L242del;A243del; L244del; R246I; A701V; T20N; P26S; D138Y; R190S; H655Y; T1027I;S13I; W152C; L452R; R346T; P384L; L452M; F456A; F456K; F456V; E484P;K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; P499H; P499S;G502V; T748K; A522S; V1176F; T859N; S247del; Y248del; L249del; T250del;P251del; G252del; R246del; S247del; Y248del; L249del; T250del; P251del;G252del; G75V; T76I; G75V; T76I; D950N; P681R; E154K; G769V; S254F;Q613H; F157L; F157del; R158del; Q957R; D253G; T95I; F888L; Q677H; A67V;Q414K; N450K; V483A; G669S; T732A; Q949R; Q1071H; E1092K; H1101Y;N1187D; W258L; V70F; T19R; T19I; Y144T; Y145S; ins145N; R346K; R346S;V126A; H245Y; ins214TDR; S12F; W152R; A899S; G142D; E156G; K558N; P9L;C136F; Y449H; L24del; P25del; P26del; A27S; V213G; S371F; T376A; D405N;D253N; Y144S; 1210T; D936N; S939F; W152L; T20I; R357K; D796H; Y145H;R682del; R683del; A684del; R685del; A701V; V143del, Y144del, Y145del,Y145N; N211del, L212del, L212I, ins214EPE, E241del, G339D, S371L, S373P,S375F, N440K, G496S, Q498R, Y505H; T547K, D614G, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F or Q52R (relative to the sequence ofSEQ ID NO: 1)

In a preferred embodiment there is provided a RNA comprising at leastone coding sequence encoding at least one SARS-CoV-2 spike protein or animmunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to H69; V70; A222;Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458;A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499;T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118;L18; D80; D215; L242; A243; L244; R246; A701; T20; P26; D138; R190;H655; T1027; S13; W152; L452; R346; P384; G447; G502; T748; A522; V1176;T859; S247; Y248; L249; T250; P251; G252; G75; T76; D950; E154; G769;S254; Q613; F157; R158; Q957; D253; T95; F888; Q677; A67; 0414; N450;V483; G669; T732; Q949; Q1071; E1092; H1101; N1187; W258; T19; V126;H245; S12; A899; G142; E156; K558; and/or Q52 relative to the sequenceof SEQ ID NO: 1, wherein the RNA comprises at least one heterologousuntranslated region. In certain aspects the RNA does not comprise a 3′UTR comprising the sequence of SEQ ID NO: 268. In certain aspects, theRNA comprises a 3′ UTR comprising the sequence of SEQ ID NO: 268.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises at least one amino acid substitution, deletion or insertion ata position corresponding to H69del; V70del; A222V; Y453F; S477N; 1692V;R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A;L455F; F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477T;T478I; T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; G485S,F486L; N487I; Y489H; F490S; F490L; Q493L; Q493K; S494P; S494L; P499L;T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; Q506H;Y144del; A570D; P681H; T716I; S982A; D1118H; L18F; D80A; D215G; L242del;A243del; L244del; L242del; A243del; L244del; R246I; A701V; T20N; P26S;D138Y; R190S; H655Y; T1027I; S13I; W152C; L452R; R346T; P384L; L452M;F456A; F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y;Q493R; 5494A; P499H; P499S; G502V; T748K; A522S; V1176F; T859N; S247de1;Y248del; L249del; T250del; P251del; G252del; R246del; S247del; Y248del;L249del; T250del; P251del; G252del; G75V; T76I; G75V; T76I; D950N;P681R; E154K; G769V; S254F; Q613H; F157L; F157del; R158del; Q957R;D253G; T95I; F888L; Q677H; A67V; Q414K; N450K; V483A; G669S; T732A;Q949R; Q1071H; E1092K; H1101Y; N1187D; W258L; V70F; T19R; Y144T; Y145S;ins145N; R346K; R346S; V126A; H245Y; ins214TDR; S12F; W152R; A899S;G142D; E156G; K558N; and/or Q52R relative to the sequence of SEQ ID NO:1.

In certain embodiments there is provided a RNA comprising at least onecoding sequence encoding at least one SARS-CoV-2 spike protein or animmunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to H69; V70; A222;Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458;A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499;T500; N501; V503; G504; Y505; and/or Q506 relative to the sequence ofSEQ ID NO: 1. Thus, in some embodiments the SARS-CoV-2 spike proteincomprises at least one amino acid substitution, deletion or insertion ata position corresponding to H69del; V70del; A222V; Y453F; S477N; 1692V;R403K; K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A;L455F; F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477T;T478I; T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; G485S,F486L; N487I; Y489H; F490S; F490L; Q493L; Q493K; S494P; S494L; P499L;T500I; N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; and/orQ506H relative to the sequence of SEQ ID NO: 1.

In a further embodiment there is provided a RNA comprising at least onecoding sequence encoding at least one SARS-CoV-2 spike protein or animmunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to H69; V70; A222;Y453; S477; I692; R403; K417; N437; N439; V445; G446; L455; F456; K458;A475; G476; T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499;T500; N501; V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118;L18; D80; D215; L242; A243; L244; R246; A701; T20; P26; D138; R190;H655; T1027; S13; W152; L452; R346; P384; G447; G502; T748; A522; orV1176 relative to the sequence of SEQ ID NO: 1. Thus, in someembodiments the SARS-CoV-2 spike protein comprises at least one aminoacid substitution, deletion or insertion at a position corresponding toH69del; V70del; A222V; Y453F; S477N; 1692V; R403K; K417N; N437S; N439K;V445A; V445I; V445F; G446V; G446S; G446A; L455F; F456L; K458N; A475V;G476S; G476A; S477I; S477R; S477G; S477T; T478I; T478K; T478R; T478A;E484Q; E484K; E484A; E484D; G485R; G485S, F486L; N487I; Y489H; F490S;F490L; Q493L; Q493K; S494P; S494L; P499L; T500I; N501Y; N501T; N501S;V503F; V503I; G504D; Y505W; Q506K; Q506H; Y144del; A570D; P681H; T716I;S982A; D1118H; L18F; D80A; D215G; L242del; A243del; L244del; L242del;A243del; L244del; R246I; A701V; T20N; P26S; D138Y; R190S; H655Y; T1027I;S13I; W152C; L452R; R346T; P384L; L452M; F456A; F456K; F456V; E484P;K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R; S494A; P499H; P499S;G502V; T748K; A522S; and/or V1176F relative to the sequence of SEQ IDNO: 1.

In a further preferred embodiment, there is provided a RNA comprising atleast one coding sequence encoding at least one SARS-CoV-2 spike proteinor an immunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to T859; R246; S247;Y248; L249; T250; P251; G252; G75; T76; D950; E154; G769; S254; Q613;F157; Q957; D253; T95; F888; Q677; A67; Q414; N450; V483; G669; T732;Q949; Q1071; E1092; H1101; N1187; F157; R158; W258; T19; H245; S12;A899; G142; E156; K558 and/or Q52 relative to the sequence of SEQ IDNO: 1. Thus, in some embodiments the SARS-CoV-2 spike protein comprisesat least one amino acid substitution, deletion or insertion at aposition corresponding to T859N; S247del; Y248del; L249del; T250del;P251del; G252del; R246del; S247del; Y248del; L249del; T250del; P251del;G252del; G75V; T76I; G75V; T76I; D950N; P681R; E154K; G769V; S254F;Q613H; F157L; Q957R; D253G; T95I; F888L; Q677H; A67V; Q414K; N450K;V483A; G669S; T732A; Q949R; Q1071 H; E1092K; H1101Y; N1187D; F157del;R158del; W258L; V70F; T19R; Y144T; Y145S; ins145N; R346K; R346S; V126A;H245Y; ins214TDR; 512F; W152R; A899S; G142D; E156G; K558N and/or Q52Rrelative to the sequence of SEQ ID NO: 1.

In still a further embodiment there is provided an RNA comprising atleast one coding sequence encoding at least one SARS-CoV-2 spike proteinor an immunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to D614; H49; V367;P1263; V483; S939; S943; L5; L8; S940; C1254; Q239; M153; V1040; A845;Y145; A831; and/or M1229 relative to the sequence of SEQ ID NO: 1. Thus,in some embodiments the SARS-CoV-2 spike protein comprises at least oneamino acid substitution, deletion or insertion at a positioncorresponding to D614G; H49Y; V367F; P1263L; V483A; S939F; S943P; L5F;L8V; S940F; C1254F; Q239K; M153T; V1040F; A845S; Y145H; A831V; and/orM1229I relative to the sequence of SEQ ID NO: 1.

In still a further embodiment there is provided an RNA comprising atleast one coding sequence encoding at least one SARS-CoV-2 spike proteinor an immunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitutionor deletion at a position corresponding to H69; V70; A222; Y453; S477;I692; R403; K417; N437; N439; V445; G446; L455; F456; K458; A475; G476;T478; E484; G485, F486; N487; Y489; F490; Q493; S494; P499; T500; N501;V503; G504; Y505; Q506; Y144; A570; P681; T716; S982; D1118; L18; D80;D215; L242; A243; L244; R246; A701; T20; P26; D138; R190; H655; T1027;S13; W152; L452; R346; P384; G447; G502; T748; A522; V1176; T859; S247;Y248; L249; T250; P251; G252; G75; T76; D950; E154; G769; S254; Q613;F157; Q957; D253; T95; F888; Q677; A67; Q414; N450; V483; G669; T732;Q949; Q1071; E1092; H1101; N1187 and/or Q52 relative to the sequence ofSEQ ID NO: 1. Thus, in some embodiments the SARS-CoV-2 spike proteincomprises at least one amino acid substitution or deletion at a positioncorresponding to H69del; V70del; A222V; Y453F; S477N; 1692V; R403K;K417N; N437S; N439K; V445A; V445I; V445F; G446V; G446S; G446A; L455F;F456L; K458N; A475V; G476S; G476A; S477I; S477R; S477G; S477T; T478I;T478K; T478R; T478A; E484Q; E484K; E484A; E484D; G485R; G485S, F486L;N487I; Y489H; F490S; F490L; Q493L; Q493K; S494P; S494L; P499L; T500I;N501Y; N501T; N501S; V503F; V503I; G504D; Y505W; Q506K; Q506H; Y144del;A570D; P681H; T716I; S982A; D1118H; L18F; D80A; D215G; L242del; A243del;L244del; L242del; A243del; L244del; R246I; A701V; T20N; P26S; D138Y;R190S; H655Y; T1027I; S13I; W152C; L452R; R346T; P384L; L452M; F456A;F456K; F456V; E484P; K417T; G447V; L452Q; A475S; F486I; F490Y; Q493R;S494A; P499H; P499S; G502V; T748K; A522S; V1176F; T859N; S247del;Y248del; L249del; T250del; P251del; G252del; R246del; S247del; Y248del;L249del; T250del; P251del; G252del; G75V; T76I; G75V; T76I; D950N;P681R; E154K; G769V; S254F; Q613H; F157L; F157del; R158del; Q957R;D253G; T95I; F888L; Q677H; A67V; Q414K; N450K; V483A; G669S; T732A;Q949R; Q1071H; E1092K; H1101Y; N1187D; W258L; V70F; T19R; Y144T; Y145S;R346K; R346S; V126A; H245Y; 512F; W152R; A899S; G142D; E156G; K558N;and/or Q52R.

In a preferred embodiment there is provided an RNA comprising at leastone coding sequence encoding at least one SARS-CoV-2 spike protein or animmunogenic fragment or immunogenic variant thereof, wherein theSARS-CoV-2 spike protein comprises at least one amino acid substitution,deletion or insertion at a position corresponding to T19I; L24del;P25del; P26del; A27S; A67V; H69del; V70del; T95I; G142D; V143del;Y144del; Y145del; N211del; L212I; V213G; ins214EPE; G339D; S371L; S371F;S373P; S375F; T376A; D405N; K417N; N440K; G446S; S477N; T478K; E484A;Q493R; G496S; Q498R; N501Y; Y505H; T547K; D614G; H655Y; N679K; P681H;A701V; N764K; D796Y; N856K; Q954H; N969K; L981F relative to the sequenceof SEQ ID NO: 1. Thus, in some embodiments the SARS-CoV-2 spike proteincomprises at least one amino acid substitution, deletion or insertion ata position corresponding to T19I; L24del; P25del; P26del; A27S; A67V;H69del; V70del; T95I; G142D; V143del; Y144del; Y145del; N211del; L212I;V213G; ins214EPE; G339D; S371L; S371F; S373P; S375F; T376A; D405N;K417N; N440K; G446S; S477N; T478K; E484A; Q493R; G496S; Q498R; N501Y;Y505H; T547K; D614G; H655Y; N679K; P681H; A701V; N764K; D796Y; N856K;Q954H; N969K; L981F relative to the sequence of SEQ ID NO: 1. In certainembodiments the SARS-CoV-2 spike protein is 90% identical to the aminoacid sequence of SEQ ID NO: 10 and comprises at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 of the aminoacid substitutions, deletions or insertions selected from the groupconsisting of T19I; L24del; P25del; P26del; A27S; A67V; H69del; V70del;T95I; G142D; V143del; Y144del; Y145del; N211del; L212I; V213G;ins214EPE; G339D; S371L; S371F; S373P; S375F; T376A; D405N; K417N;N440K; G446S; S477N; T478K; E484A; Q493R; G496S; Q498R; N501Y; Y505H;T547K; D614G; H655Y; N679K; P681H; A701V; N764K; D796Y; N856K; Q954H;N969K; L981F relative to the sequence of SEQ ID NO: 1.

In yet a further embodiment there is provided an RNA comprising at leastone coding sequence encoding at least one SARS-CoV-2 spike protein or animmunogenic fragment thereof, wherein the SARS-CoV-2 spike proteincomprises at least one amino acid substitution, insertion or deletioncorresponding to A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at a position located in the RBD domain (aminoacid position aa 319 to aa 541; amino acid positions according toreference SEQ ID NO: 1) or the CND domain (amino acid position aa 329 toaa 529; amino acid positions according to reference SEQ ID NO: 1).Without wishing to be bound to theory, amino acid substitutions ormutations in the CND domain may help new emerging SARS-CoV-2 variants toevade antibody detection of some types of antibodies induced in subjectsvaccinated with first generation vaccines (designed against the originalSARS-CoV-2 strain) or induced in subjects after infection with theoriginal SARS-CoV-2 strain.

Accordingly, in preferred embodiments, the first aspect of the inventionrelates to an RNA comprising at least one coding sequence encoding atleast one antigenic peptide or protein from a SARS-CoV-2 spike proteinor an immunogenic fragment or immunogenic variant thereof, wherein theRNA comprises at least one heterologous untranslated region (UTR) andwherein the SARS-CoV-2 spike protein comprises at least one amino acidsubstitution at position located in the RBD domain (amino acid positionaa 319 to aa 541; amino acid positions according to reference SEQ IDNO: 1) or the CND domain (amino acid position aa 329 to aa 529 aminoacid positions according to reference SEQ ID NO: 1).

In certain preferred embodiments, the SARS-CoV-2 spike protein comprisesan amino acid substitution, insertion or deletion in at least one of thefollowing positions: R346; V367, P384; R403; K417; N437; N439; V445;G446; G447; N450; L452; Y453; L455; F456; A475; G476; S477; T478; E484;G485; F486; N487; Y489; F490; Q493; S494; P499; T500; N501; G502; V503;G504; Y505; Q506; A522 (amino acid positions according to reference SEQID NO: 1).

Accordingly, in certain preferred embodiments, the first aspect of theinvention relates to an RNA comprising at least one coding sequenceencoding at least one antigenic peptide or protein from a SARS-CoV-2spike protein or an immunogenic fragment or immunogenic variant thereof,wherein the SARS-CoV-2 spike protein comprises at least one amino acidsubstitution at positions selected from K417; L452; T478; E484; N501and/or P681 (amino acid positions according to reference SEQ ID NO: 1)and wherein the RNA comprises at least one heterologous untranslatedregion (UTR).

Without wishing to be bound to theory, an amino acid substitution atposition E484 may help SARS-CoV-2 virus variants to evade antibodydetection of some types of antibodies induced in subjects vaccinatedwith first generation vaccines (designed against the original SARS-CoV-2strain) or induced in subjects after infection with the originalSARS-CoV-2 strain. A mutation/substitution in N501 occurs near the topof the coronavirus spike, where it may alter the shape of the protein,which may help to evade some types of coronavirus antibodies. SuchSARS-CoV-2 are called SARS-CoV-2 E484 variants throughout the presentinvention and include e.g. SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2B.1.617 (India), P.1 (Brazil) or B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5).

Accordingly, in some embodiments, it may be advantageous that the RNA ofthe invention provides a SARS-CoV-2 spike protein comprising asubstitution in position E484 to allow the induction of efficient immuneresponses against virus SARS-CoV-2 E484 variants.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position E484, wherein the amino acids E484is substituted with K, P, Q, A, or D (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises aE484K, E484P, E484Q, E484A, E484D amino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises an amino acid substitution at position E484, wherein the aminoacids E484 is substituted with K or Q (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises a E484Kor E484Q amino acid substitution. In certain preferred embodiments aSARS-CoV-2 spike protein comprises a E484K amino acid substitution.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position N501, wherein the amino acids N501is substituted with a different amino acid (amino acid positionsaccording to reference SEQ ID NO: 1).

Without wishing to be bound to theory, an amino acid substitution atposition N501 may help SARS-CoV-2 virus variants to evade antibodydetection of some types of antibodies induced in subjects vaccinatedwith first generation vaccines (designed against the original SARS-CoV-2strain) or induced in subjects after infection with the originalSARS-CoV-2 strain. A mutation/substitution in N501 occurs near the topof the coronavirus spike, where it may alter the shape of the protein,which may help to evade some types of coronavirus antibodies. SuchSARS-CoV-2 are called SARS-CoV-2 N501 variants throughout the presentinvention and include e.g. SARS-CoV-2 B.1.351 (South Africa), SARS-CoV-2B.1.1.7 (UK), P.1 (Brazil), or B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5).

Accordingly it may be advantageous that the RNA of the inventionprovides a SARS-CoV-2 spike protein comprising a substitution inposition N501 to allow the induction of efficient immune responsesagainst virus SARS-CoV-2 N501 variants.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position N501, wherein the amino acids N501is substituted with Y, T, S (amino acid positions according to referenceSEQ ID NO: 1). Accordingly, the antigenic peptide or protein selectedfrom or derived from SARS-CoV-2 spike protein comprises a N501Y, N501T,N501S amino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises an amino acid substitution at position N501, wherein the aminoacids N501 is substituted with Y (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises a N501Yamino acid substitution.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position K417, wherein the amino acids K417is substituted with a different amino acid (amino acid positionsaccording to reference SEQ ID NO: 1).

Without wishing to be bound to theory, an amino acid substitution atposition K417 may help SARS-CoV-2 virus variants to evade antibodydetection of some types of antibodies induced in subjects vaccinatedwith vaccines designed against the original SARS-CoV-2 strain using SEQID NO:1 or induced in subjects after infection with the originalSARS-CoV-2 strain comprising SEQ ID NO:1. A mutation/substitution inK417 occurs near the top of the coronavirus spike, where it may alterthe shape of the protein, which may help to evade some types ofcoronavirus antibodies. Such SARS-CoV-2 are called SARS-CoV-2 K417variants throughout the present invention and include e.g. SARS-CoV-2B.1.351 (South Africa), SARS-CoV-2 B.1.1.7 (UK), P.1 (Brazil), AY.1/AY.2or or B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).

Accordingly it may be advantageous that the RNA of the inventionprovides a SARS-CoV-2 spike protein comprising a substitution inposition K417 to allow the induction of efficient immune responsesagainst virus SARS-CoV-2 K417 variants.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position K417, wherein the amino acids N501is substituted with S, T, Q or N (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises aK417S, K417T, K417Q or K417N amino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises an amino acid substitution at position N501, wherein the aminoacids K417 is substituted with T or N (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises a K417Tor K417N amino acid substitution. In certain preferred embodiments theantigenic peptide or protein selected from or derived from SARS-CoV-2spike protein comprises a K417N amino acid substitution.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position L452, wherein the amino acids L452is substituted with a different amino acid (amino acid positionsaccording to reference SEQ ID NO: 1).

Without wishing to be bound to theory, an amino acid substitution atposition L452 may help SARS-CoV-2 virus variants to evade antibodydetection of some types of antibodies induced in subjects vaccinatedwith vaccines designed against the original SARS-CoV-2 strain based onSEQ ID NO:1 or induced in subjects after infection with the originalSARS-CoV-2 strain having SEQ ID NO:1. A mutation/substitution in L452occurs near the top of the coronavirus spike, where it may alter theshape of the protein, which may help to evade some types of coronavirusantibodies. Such SARS-CoV-2 are called SARS-CoV-2 L452 variantsthroughout the present invention and include e.g. SARS-CoV-2 B.1.617.1(India), SARS-CoV-2 B.1.617.2 (India), or SARS-CoV-2 B.1.617.3 (India).

Accordingly it may be advantageous that the RNA of the inventionprovides a SARS-CoV-2 spike protein comprising a substitution inposition L452 to allow the induction of efficient immune responsesagainst virus SARS-CoV-2 L452 variants.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position L452, wherein the amino acids L452is substituted with R or Q (amino acid positions according to referenceSEQ ID NO: 1). Accordingly, the antigenic peptide or protein selectedfrom or derived from SARS-CoV-2 spike protein comprises an L452R orL452Q amino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises an amino acid substitution at position L452, wherein the aminoacids L452 is substituted with R (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises a L452Ramino acid substitution.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at a position located in the furin cleavage site(amino acid position aa 681 to 685; amino acid positions according toreference SEQ ID NO: 1). That sequence stretch (PRRAR in SEQ ID NO: 1)is believed to serve as a recognition site for furin cleavage.

Without wishing to be bound to theory, amino acid substitutions ormutations in the furin cleavage site may help new emerging SARS-CoV-2variants to have increased membrane fusion and thus cause increasedtransmissibility.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position P681 in the furin cleavage site.Suitably, the amino acids P681 is substituted with a different aminoacid (amino acid positions according to reference SEQ ID NO: 1),preferably an amino acid that improves furin cleavage. Such SARS-CoV-2are called SARS-CoV-2 P681 variants throughout the present invention andinclude e.g. SARS-CoV-2 B.1.617.1 (India), SARS-CoV-2 B.1.617.2 (India),or SARS-CoV-2 B.1.617.3 (India), SARS-CoV-2 B.1.1.7 (UK), SARS-CoV-2A.23.1 (Uganda), or B.1.1.529 (Omicron, South Africa) (includingBA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5).

Accordingly it may be advantageous that the RNA of the inventionprovides a SARS-CoV-2 spike protein comprising a substitution inposition P681 to allow the induction of efficient immune responsesagainst virus SARS-CoV-2 P681 variants.

In preferred embodiments, the SARS-CoV-2 spike protein comprises anamino acid substitution at position P681, wherein the amino acids P681is substituted with R or H (amino acid positions according to referenceSEQ ID NO: 1). Accordingly, the antigenic peptide or protein selectedfrom or derived from SARS-CoV-2 spike protein comprises an P681R orP681H amino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike proteincomprises an amino acid substitution at position P681, wherein the aminoacids P681 is substituted with R (amino acid positions according toreference SEQ ID NO: 1). Accordingly, the antigenic peptide or proteinselected from or derived from SARS-CoV-2 spike protein comprises a P681Ramino acid substitution.

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises an amino acidsubstitution at position L452 as defined herein, preferably L452R, andan amino acid substitution at position P681 as defined herein,preferably P681R (amino acid positions according to reference SEQ ID NO:1).

In another particularly preferred embodiment, the SARS-CoV-2 spikeprotein that is encoded by the RNA of the invention comprises an aminoacid substitution at position L452 as defined herein, preferably L452R,and an amino acid substitution at position P681 as defined herein,preferably P681R (amino acid positions according to reference SEQ ID NO:1). In further preferred embodiment, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises an amino acidsubstitution at position L452 as defined herein, preferably L452R, anamino acid substitution at position P681 as defined herein, preferablyP681R and at position D614 as defined herein, preferably D614G, (aminoacid positions according to reference SEQ ID NO: 1).

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises an amino acidsubstitution at position N501 as defined herein, preferably N501Y, andan amino acid substitution at position E484 as defined herein,preferably E484K (amino acid positions according to reference SEQ ID NO:1).

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises an amino acidsubstitution at position L452 as defined herein, preferably L452R, andan amino acid substitution at position E484 as defined herein,preferably E484Q (amino acid positions according to reference SEQ ID NO:1).

In preferred embodiments, the SARS-CoV-2 spike protein comprises, inaddition to the substitutions defined above (at positions E484, N501,L452 and optionally P681), at least one, in particular 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 additional amino acid substitution, insertion or deletionselected from List 1A or List 1B.

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises an amino acidsubstitution or deletion at position H69 as defined herein, preferablyH69del, and an amino acid substitution or deletion at position V70 asdefined herein, preferably V70del (amino acid positions according toreference SEQ ID NO: 1). In further preferred embodiment, the SARS-CoV-2spike protein that is encoded by the RNA of the invention comprises adeletion at both H69 and V70.

In preferred embodiments, the SARS-CoV-2 spike protein that is encodedby the RNA of the invention comprises at least one further amino acidsubstitution or deletion selected from the following SARS-CoV-2isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US),B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India),AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, SouthAfrica), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429(Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czechrepublic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1(Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1(Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu, Columbia).

In preferred embodiments, the SARS-CoV-2 spike protein that is encodedby the RNA of the invention comprises amino acid substitutions ordeletions selected from (relative to SEQ ID NO: 1):

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v1);

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,L981F (SA, BA.1_v0);

K986P, V987P, A67V, T95I, G339D, S371L, S373P, S375F, S477N, T478K,E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,P681H, D796Y, N856K, Q954H, N969K, L981F (SA, B.1.1.529);

K986P, V987P, T19I, L24del, P25del, P26del, A27S, G142D, V213G, G339D,S371F, S373P, S375F, T376A, D405N, S477N, T478K, E484A, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, D796Y, Q954H, N969K (SA,BA.2);

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, N440K,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA,BA.1_v2);

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, D796Y, N856K, Q954H, N969K, L981F (SA, BA.1_v3);

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, L981F (SA,BA.1_v4);

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (SA,BA.1_v5);

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V; (SA; B.1.351)

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V; (SA; B.1.351)

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, andT1027I; (Brazil; P1)

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y,T1027I, and V1176F; (Brazil P1)

L452R, P681R, and D614G; (B.1.617.1; India)

L452R, E484Q, P681R, E154K, D614G, and Q1071H; (B.1.617.2; India)

L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N;(B.1.617.2; India)

T19R, L452R, E484Q, D614G, P681R and D950N; (B.1.617.3; India)

G75V, T76I, S247del, Y248del, L249del, T250del, P251del, G252del,D253del, L452Q, F490S, D614G, and T859N; (C.37.1; Peru)

T95I, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N; (B.1.1.621)

T95I, Y144T, Y145S, ins145N, R346K, E484K, N501Y, D614G, P681H, andD950N; (B.1.1.621)

H69del, V70del, Y144del, E484K, N501Y, A570D, D614G, P681H, T716I,S982A, and D1118H; (B.1.1.7—E484K)

S13I, W152C, L452R, and D614G; (B.1.429)

L452R; and D614G; (B.1.429)

H69del; V70del; N439K; D614G; (B.1.258)

T95I; E484K; D614G; and A701V; (B.1.526)

L5F, T95I, D253G, E484K, D614G, and A701V; (B.1.526)

L5F, T95I, D253G, S477N, D614G, and Q957R; (B.1.526)

F157L; V367F; Q613H; and P681R (A.23.1)

S254F; D614G; P681R; and G769V (A.23.1)

T478K; D614G; P681H; and T732A (B.1.1.519; Mexico)

P26S, H69del, V70del, V126A, Y144del, L242del, A243del, L244del, H245Y,S477N, E484K, D614G, P681H, T1027I and D1118H; (B.1.620; Africa)

ins214TDR, Q414K, N450K, D614G, and T716I; (B.1.214.2)

S12F, H69del, V70del, W152R, R346S, L452R, D614G, Q677H and A899S;(C.36.3; Thailand)

E484K, D614G and V1176F; (P2)

Q52R; A67V; H69del; V70del; F157del; R158del; E484K; D614G; Q677H andF888L; (B.1.525)

Q52R; A67V; H69del; V70del; Y144del; E484K; D614G; Q677H and F888L;(B.1.525)

A67V; H69del; V70del; Y144del; E484K; D614G; Q677H and F888L; (B.1.525)

T19R; T95I; G142D, E156G, F157del; R158del; W258L; K417N; L452R; T478K;K558N, D614G; P681R; and D950N; (AY.1)

T19R; V70F; G142D, E156G, F157del; R158del; A222V, K417N; L452R; T478K;D614G; P681R; and D950N; (AY.2)

T19R; T95I; F157del; R158del; W258L; K417N; L452R; T478K; D614G; P681R;and D950N; or (AY.1)

T19R; V70F; F157del; R158del; A222V; K417N; L452R; T478K; D614G; P681R;and D950N; (AY.2)

H69del, V70del and D614G;

D614G and M1229;

A222V and D614G;

S477N and D614G;

N439K and D614G;

H69del, V70del, Y453F, D614G and I692I;

Y453F and D614G;

D614G and I692V;

H69del, V70del, A222V, Y453F, D614G and I692I;

N501Y and D614G;

K417N; E484K; N501Y and D614G; or

E484K and D614G.

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises the following aminoacid substitutions or deletions (relative to SEQ ID NO: 1):

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V;

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, andT1027I;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y,T1027I, and V1176F;

L452R, P681R, and D614G;

L452R, E484Q, P681R, E154K, D614G, and Q1071H; or

L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.

In even more preferred embodiments, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises the following amino acidsubstitutions or deletions (relative to SEQ ID NO: 1):

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V; or

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V.

In further particularly preferred embodiments, the SARS-CoV-2 spikeprotein that is encoded by the RNA of the invention comprises thefollowing amino acid substitutions or deletions (relative to SEQ ID NO:1):

L452R, P681R, and D614G;

L452R, E484Q, P681R, E154K, D614G, and Q1071H;

L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N; or

T19R, L452R, E484Q, D614G, P681R and D950N.

In even more preferred embodiments, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 1, 10, 22738, 22740, 22742,22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964,27087-27109, 28540-28588, 28917-28920, or an immunogenic fragment orimmunogenic variant of any of these. Thus, in some embodiments, theSARS-CoV-2 spike protein is at least 95%, identical to any one of SEQ IDNOs: 1, 10, 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752,22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or28917-28920. In certain embodiments, the SARS-CoV-2 spike protein isidentical to any one of SEQ ID NOs: 22738, 22740, 22742, 22744, 22746,22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109,28540-28588, or 28917-28920. Further information regarding said aminoacid sequences is also provided in Table 1, and under <223> identifierof the ST25 sequence listing of respective sequence SEQ ID NOs.

In some embodiments, a fragment of a spike protein (S) as defined hereinmay be encoded by the RNA of the invention, wherein said fragment may beN-terminally truncated, lacking the N-terminal amino acids 1 to up to100 of the full length SARS-CoV-2 variant protein and/or wherein saidfragment may be C-terminally truncated, lacking the C-terminal aminoacids (aa) 531 to up to aa 1273 of the full length SARS-CoV-2 variantprotein. Such “fragment of a spike protein (S)” may additionallycomprise amino acid substitutions (as described herein) and mayadditionally comprise at least one heterologous peptide or proteinelement (as described herein). In preferred embodiments, a fragment of aspike protein (S) may be C-terminally truncated, thereby lacking theC-terminal transmembrane domain (that is, lacking aa 1212 to aa 1273 orlacking aa 1148 to aa 1273) (amino acid positions according to referenceSEQ ID NO: 1).

In other embodiments, the encoded spike protein (S) derived fromSARS-CoV-2 lacks the transmembrane domain (TM) (amino acid position aa1212 to aa 1273 according to reference SEQ ID NO: 1). In embodiments,the encoded spike protein (S) derived from SARS-CoV-2 lacks an extendedpart of the transmembrane domain (TMflex) (amino acid position aa 1148to aa 1273 according to reference SEQ ID NO: 1). Without wishing tobeing bound to theory, a spike protein (S) lacking the transmembranedomain (TM or TMflex) as defined herein could be suitable for a vaccine,as such a protein would be soluble and not anchored in the cellmembrane. A soluble protein may therefore be produced (that istranslated) in higher concentrations upon administration to a subject,leading to improved immune responses.

Without wishing to being bound to theory, RBD (aa 319 to aa 541) and CND(aa 329 to aa 529) domains, as referenced for amino acid positions withSEQ ID NO:1, may be crucial for immunogenicity. Both regions are locatedat the S1 fragment of the spike protein. Accordingly, it may be suitablein the context of the invention that the antigenic peptide or proteincomprises or consists of an S1 fragment of the spike protein or animmunogenic fragment or immunogenic variant thereof. Suitably, such anS1 fragment may comprise at least an RBD and/or a CND domain as definedabove. In certain embodiments the SARS-CoV-2 spike protein CND domainthat is encoded by the RNA of the invention comprises or consists of atleast one of the amino acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 27051-27086 or animmunogenic fragment or immunogenic variant of any of these. Thus, insome embodiments, the SARS-CoV-2 spike protein CND domain is at least95%, identical to any one of SEQ ID NOs: 27051-27086. In certainembodiments, the SARS-CoV-2 spike protein CND domain is identical to anyone of SEQ ID NOs: 27051-27086. Further information regarding said aminoacid sequences is also provided in Table 1, and under <223> identifierof the ST25 sequence listing of respective sequence SEQ ID NOs.

In preferred embodiments, the encoded at least one antigenic peptide orprotein comprises or consists of a receptor-binding domain (RBD; aa 319to aa 541), wherein the RBD comprises or consists of a spike proteinfragment, or an immunogenic fragment or immunogenic variant thereof. Incertain embodiments the SARS-CoV-2 spike protein RBD domain that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 27007-27046 or an immunogenicfragment or immunogenic variant of any of these. Thus, in someembodiments, the SARS-CoV-2 spike protein RBD domain is at least 95%,identical to any one of SEQ ID NOs: 27007-27046. In certain embodiments,the SARS-CoV-2 spike protein RBD domain is identical to any one of SEQID NOs: 27007-27046. Further information regarding said amino acidsequences is also provided in Table 1, and under <223> identifier of theST25 sequence listing of respective sequence SEQ ID NOs.

In further preferred embodiments, the encoded at least one antigenicpeptide or protein comprises or consists of a truncated receptor-bindingdomain (truncRBD; aa 334 to aa 528), wherein the RBD comprises orconsists of a spike protein fragment, or an immunogenic fragment orimmunogenic variant thereof.

Such “fragment of a spike protein (S)” (RBD; aa 319 to aa 541 ortruncRBD, aa 334 to aa 528), may additionally comprise amino acidsubstitutions (as described herein) and may additionally comprise atleast one heterologous peptide or protein element (as described herein).

In particularly preferred embodiments, the encoded at least oneantigenic peptide or protein comprises or consists of a spike protein(S), wherein the spike protein (S) comprises or consists of a spikeprotein fragment S1, or an immunogenic fragment or immunogenic variantthereof.

In preferred embodiments, the encoded at least one antigenic peptide orprotein comprises a spike protein fragment S1, and lacks at least 70%,80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% of spike protein fragment S2 (aa682 to aa 1273). Such embodiments may be beneficial, as the S1 fragmentcomprises neutralizing epitopes.

Without wishing to being bound to theory, it may be suitable that theantigenic peptide or protein comprises or consists of spike proteinfragment S1 and (at least a fragment of) spike protein fragment S2,because the formation of an immunogenic spike protein may be promoted.

Accordingly, in particularly preferred embodiments, the encoded at leastone antigenic peptide or protein comprises or consists of a spikeprotein (S), wherein the spike protein (S) comprises or consists of aspike protein fragment S1 or an immunogenic fragment or immunogenicvariant thereof, and spike protein fragment S2 or an immunogenicfragment or immunogenic variant thereof.

In alternative preferred embodiments, the encoded at least one antigenicpeptide or protein comprises or consists of a full-length spike proteinor an immunogenic fragment or immunogenic variant of any of these.

The term “full length spike protein” has to be understood as a spikeprotein derived from a SARS-CoV-2 having an amino acid sequencecorresponding to essentially the full spike protein. Accordingly, a“full length spike protein” may comprise aa 1 to aa 1273 (referenceprotein: SEQ ID NO: 1). Accordingly, a full length spike protein maytypically comprise a secretory signal peptide, a spike protein fragmentS1, a spike protein fragment S2, a receptor binding domain (RBD), and acritical neutralisation domain CND, and a transmembrane domain. Notably,also variants that comprise certain amino acid substitutions (e.g. forallowing pre-fusion stabilization of the S protein) or natural occurringamino acid deletions are encompassed by the term “full length spikeprotein”.

In particularly preferred embodiments, the spike protein (S) that isencoded by the RNA of the first aspect is designed or adapted tostabilize the antigen in pre-fusion conformation. A pre-fusionconformation is particularly advantageous in the context of an efficientcoronavirus vaccine, as several potential epitopes for neutralizingantibodies may merely be accessible in said pre-fusion proteinconformation. Furthermore, remaining of the protein in the pre-fusionconformation is aimed to avoid immunopathological effects, like e.g.enhanced disease and/or antibody dependent enhancement (ADE).

In preferred embodiments, administration of the RNA (or a composition orvaccine) encoding pre-fusion stabilized spike protein to a subjectelicits spike protein neutralizing antibodies and does not elicitdisease-enhancing antibodies. In particular, administration of a nucleicacid (or a composition or vaccine) encoding pre-fusion stabilized spikeprotein to a subject does not elicit immunopathological effects, likee.g. enhanced disease and/or antibody dependent enhancement (ADE).

Accordingly, in preferred embodiments, the RNA of the inventioncomprises at least one coding sequence encoding at least one antigenicpeptide or protein that is selected or is derived from a SARS-CoV-2spike protein (S), wherein the SARS-CoV-2 spike protein (S) is apre-fusion stabilized spike protein (S_stab). Suitably, said pre-fusionstabilized spike protein comprises at least one pre-fusion stabilizingmutation.

The term “pre-fusion conformation” as used herein relates to astructural conformation adopted by the ectodomain of the SARS-CoV-2 Sprotein following processing into a mature SARS-CoV-2 S protein in thesecretory system, and prior to triggering of the fusogenic event thatleads to transition of the SARS-CoV-2 S to the postfusion conformation.

A “pre-fusion stabilized spike protein (S_stab)” as described hereincomprises one or more amino acid substitutions, deletions, or insertionscompared to a native SARS-CoV-2 S sequence that provide for increasedretention of the prefusion conformation compared to SARS-CoV-2 Sectodomain trimers formed from a corresponding native SARS-CoV-2 Ssequence. The “stabilization” of the prefusion conformation by the oneor more amino acid substitutions, deletions, or insertions can be, forexample, energetic stabilization (for example, reducing the energy ofthe prefusion conformation relative to the post-fusion openconformation) and/or kinetic stabilization (for example, reducing therate of transition from the prefusion conformation to the postfusionconformation). Additionally, stabilization of the SARS-CoV-2 Sectodomain trimer in the prefusion conformation can include an increasein resistance to denaturation compared to a corresponding nativeSARS-CoV-2 S sequence.

Accordingly, in preferred embodiments, the SARS-CoV-2 spike proteinincludes one or more amino acid substitutions that stabilize the Sprotein in the pre-fusion conformation, for example, substitutions thatstabilize the membrane distal portion of the S protein (including theN-terminal region) in the pre-fusion conformation.

Stabilization of the SARS-CoV-2 coronavirus spike protein may beobtained by substituting at least one amino acid at position K986 and/orV987 with amino acids that stabilize the spike protein in a prefusionconformation (amino acid positions according to reference SEQ ID NO: 1).

In preferred embodiments, the pre-fusion stabilizing mutation comprisesan amino acid substitution at position K986 and V987, wherein the aminoacids K986 and/or V987 are substituted with an amino acid selected fromA, I, L, M, F, V, G, or P (amino acid positions according to referenceSEQ ID NO: 1).

Preferably, stabilization of the prefusion conformation is obtained byintroducing two consecutive proline substitutions at residues K986 andV987 in the spike protein (amino acid positions according to referenceSEQ ID NO: 1). Accordingly, in preferred embodiments, the pre-fusionstabilized spike protein (S_stab) comprises at least one pre-fusionstabilizing mutation, wherein the at least one pre-fusion stabilizingmutation comprises the following amino acid substitutions: K986P andV987P (amino acid positions according to reference SEQ ID NO: 1).

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention is a pre-fusion stabilized spikeprotein (S_stab) comprising at least one pre-fusion stabilizing K986Pand V987P mutation and additionally comprising the following amino acidsubstitutions or deletions (amino acid positions according to referenceSEQ ID NO: 1):

E484K, N501Y, and optionally L18F, D80A, D215G, L242del, A243del,L244del, R246I, K417N, D614G, A701V;

E484K, N501Y, and optionally L18F, D80A, D215G, L242del, A243del,L244del, K417N, D614G, A701V;

N501Y, P681H, and optionally H69del, V70del, Y144del, A570D, D614G,T716I, S982A, D1118H;

N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D,D614G, T716I, S982A, D1118H;

E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T,D614G, H655Y, T1027I;

E484K, N501Y, and optionally L18F, T20N, P26S, D138Y, R190S, K417T,D614G, H655Y, T1027I, V1176F;

N501Y, P681H, E484K, and optionally H69del, V70del, Y144del, A570D,D614G, T716I, S982A, D1118H;

L452R, and optionally S13I, W152C, D614G;

L452R, D614D and optionally P681R;

L452R, D614D, P681R and optionally E484Q, E154K, Q1071H;

L452R, D614D, P681R and optionally T19R, L452R, D950N;

L452R, D614D, P681R and optionally T19R, F157del, T478K, D950N;

E484K, and optionally Q52R, A67V, H69del, V70del, delY144, D614G, Q677H,F888L;

E484K, and optionally A67V, H69del, V70del, Y144del, D614G, Q677H,F888L;

E484K, and optionally L5F, T95I, D253G, D614G, A701V;

P681R, and optionally F157L, V367F, Q613H;

P681R, and optionally S254F, D614G, G769V;

L452R, P681R, and optionally D614G;

L452R, E484Q, P681R, and optionally E154K, D614G, Q1071H;

L452R, P681R, and optionally T19R, F157del, R158del, T478K, D614G,D950N;

E484K, and optionally D614G, V1176F;

L452Q, and optionally G75V, T76I, R246del, S247del, Y248del, L249del,T250del, P251del, G252del, F490S, D614G, T859N;

K417N, and optionally P681R;

K417N, P681R, and optionally D614G;

K417N, L452R, P681R, and optionally D614G;

K417N, T478K, P681R, and optionally D614G;

K417N, D950N, P681R, and optionally D614G.

K417N, D614G, P681R, and optionally T478K;

K417N, D614G, P681R, and optionally L452R;

K417N, D614G, P681R, L452R and optionally T478K;

S247del, Y248del, L249del, T250del, P251del, G252del, D253del andoptionally D614G;

S247del, Y248del, L249del, T250del, P251del, G252del, D253del andoptionally L452Q, D614G;

H69del, V70del and optionally D614G;

H69del, V70del, E484K and optionally D614G;

H69del, V70del, N501Y and optionally D614G; or

H69del, V70del, N501Y, E484K and optionally P681H.

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention is a pre-fusion stabilized spikeprotein (S_stab) (or a fragment or variant thereof) comprising at leastone pre-fusion stabilizing K986P and V987P mutation and additionallycomprises the following amino acid substitutions or deletions (aminoacid positions according to reference SEQ ID NO: 1):

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V;

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, andT1027I;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y,T1027I, V1176F;

L452R, P681R, and D614G;

L452R, E484Q, P681R, E154K, D614G, and Q1071H; or

L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.

In particularly preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention is a pre-fusion stabilized spikeprotein (S_stab) (or a fragment or variant thereof) comprising aminoacid substitutions or deletions selected from (amino acid positionsaccording to reference SEQ ID NO: 1):

K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del,L244del, R246I, K417N, D614G, and A701V; or

K986P, V987P, E484K, N501Y, L18F, D80A, D215G, L242del, A243del,L244del, K417N, D614G, and A701V.

It has to be emphasized that in the context embodiments of the inventionany SARS-CoV-2 coronavirus spike protein as defined herein may bemutated as described above (exemplified for reference protein SEQ IDNO: 1) to stabilize the spike protein in the pre-fusion conformation.

According to various embodiments, the RNA of the invention encodes atleast one antigenic SARS-CoV-2 spike protein as defined herein and,additionally, at least one heterologous peptide or protein element.

Suitably, the at least one heterologous peptide or protein element maypromote or improve secretion of the encoded antigenic SARS-CoV-2 spikeprotein (e.g. via secretory signal sequences), promote or improveanchoring of the encoded antigenic SARS-CoV-2 spike protein in theplasma membrane (e.g. via transmembrane elements), promote or improveformation of antigen complexes (e.g. via multimerization domains orantigen clustering elements), or promote or improve virus-like particleformation (VLP forming sequence). In addition, the RNA of the firstaspect may additionally encode peptide linker elements, self-cleavingpeptides, immunologic adjuvant sequences or dendritic cell targetingsequences.

Suitable multimerization domains may be selected from the list of aminoacid sequences according to SEQ ID NOs: 1116-1167 of WO2017/081082, orfragments or variants of these sequences. Suitable transmembraneelements may be selected from the list of amino acid sequences accordingto SEQ ID NOs: 1228-1343 of WO2017/081082, or fragments or variants ofthese sequences. Suitable VLP forming sequences may be selected from thelist of amino acid sequences according to SEQ ID NOs: 1168-1227 of thepatent application WO2017/081082, or fragments or variants of thesesequences. Suitable peptide linkers may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1509-1565 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable self-cleaving peptides may be selected from the list of aminoacid sequences according to SEQ ID NOs: 1434-1508 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable immunologic adjuvant sequences may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1360-1421 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable dendritic cell (DCs) targeting sequences may be selected fromthe list of amino acid sequences according to SEQ ID NOs: 1344-1359 ofthe patent application WO2017/081082, or fragments or variants of thesesequences. Suitable secretory signal peptides may be selected from thelist of amino acid sequences according to SEQ ID NOs: 1-1115 and SEQ IDNO: 1728 of published PCT patent application WO2017/081082, or fragmentsor variants of these sequences.

In preferred embodiments, the RNA encoding at least one antigenicSARS-CoV-2 spike protein additionally encodes at least one heterologoussecretory signal sequences and/or trimerization element, and/or antigenclustering element, and/or VLP forming sequence.

Accordingly, in preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 22738, 22740, 22742, 22744,22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964,27087-27109, 28540-28588, 28917-28920 or an immunogenic fragment orimmunogenic variant of any of these. Thus in some embodiments, theSARS-CoV-2 spike protein is at least 95%, identical to any one of SEQ IDNOs: 22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754,22756, 22758, 22959-22964, 27087-27109, 28540-28588, or 28917-28920. Incertain embodiments, the SARS-CoV-2 spike protein is identical to anyone of SEQ ID NOs: 22738, 22740, 22742, 22744, 22746, 22748, 22750,22752, 22754, 22756, 22758, 22959-22964, 27087-27109, 28540-28588, or28917-28920. Further information regarding said amino acid sequences isalso provided in Table 1, and under <223> identifier of the ST25sequence listing of respective sequence SEQ ID NOs.

Accordingly, in preferred embodiments, the SARS-CoV-2 spike protein thatis encoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 27093-27095, 28552-28558 or animmunogenic fragment or immunogenic variant of any of these. Thus insome embodiments, the SARS-CoV-2 spike protein is at least 95%,identical to any one of SEQ ID NOs: 27093-27095, 28552-28558. In certainembodiments, the SARS-CoV-2 spike protein is identical to any one of SEQID NOs: 27093-27095, 28552-28558.

In further preferred embodiments, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 27095, 28552-28557 or animmunogenic fragment or immunogenic variant of any of these. Thus insome embodiments, the SARS-CoV-2 spike protein is at least 95%,identical to any one of SEQ ID NOs: 27095, 28552-28557. In certainembodiments, the SARS-CoV-2 spike protein is identical to any one of SEQID NOs: 27095, 28552-28557.

In further preferred embodiments, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 27095 or an immunogenic fragmentor immunogenic variant of any of these. Thus in some embodiments, theSARS-CoV-2 spike protein is at least 95%, identical to any one of SEQ IDNOs: 27095. In certain embodiments, the SARS-CoV-2 spike protein isidentical to any one of SEQ ID NOs: 27095.

In further preferred embodiments, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences or amino acid coding sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:23090, 23091, 22960, 22961, 28540 or an immunogenic fragment orimmunogenic variant of any of these. Thus in some embodiments, theSARS-CoV-2 spike protein is at least 95%, identical to any one of SEQ IDNOs: 23090, 23091, 22960, 22961, 28540. In certain embodiments, theSARS-CoV-2 spike protein is identical to any one of SEQ ID NOs: 23090,23091, 22960, 22961, 28540.

In still further preferred embodiments, the SARS-CoV-2 spike proteinthat is encoded by the RNA of the invention comprises or consists of atleast one of the amino acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 27096, 28545 or animmunogenic fragment or immunogenic variant of any of these. Thus insome embodiments, the SARS-CoV-2 spike protein is at least 95%,identical to any one of SEQ ID NOs: 27096, 28545. In certainembodiments, the SARS-CoV-2 spike protein is identical to any one of SEQID NOs: 27096, 28545.

In still a further preferred embodiment, the SARS-CoV-2 spike proteinthat is encoded by the RNA of the invention comprises or consists of atleast one of the amino acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 22959 or an immunogenicfragment or immunogenic variant of any of these. Thus in someembodiments, the SARS-CoV-2 spike protein is at least 95%, identical toany one of SEQ ID NOs: 22959. In certain embodiments, the SARS-CoV-2spike protein is identical to any one of SEQ ID NOs: 22959.

In a further preferred embodiment, the SARS-CoV-2 spike protein that isencoded by the RNA of the invention comprises or consists of at leastone of the amino acid sequences being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to any one of SEQ ID NOs: 28541-28544, 28917-28920 or animmunogenic fragment or immunogenic variant of any of these. Thus insome embodiments, the SARS-CoV-2 spike protein is at least 95%,identical to any one of SEQ ID NOs: 28541-28544, 28917-28920. In certainembodiments, the SARS-CoV-2 spike protein is identical to any one of SEQID NOs: 28541-28544, 28917-28920.

Preferred antigenic peptide or proteins derived from an SARS-CoV-2 asdefined herein are provided in Table 1. Therein, each row corresponds toa suitable SARS-CoV-2 spike protein construct. Column A of Table 1provides a short description of the suitable antigen constructs. ColumnB of Table 1 provides protein (amino acid) SEQ ID NOs of respectiveantigen constructs. Column C Table 1 provides SEQ ID NO of thecorresponding G/C optimized nucleic acid coding sequences (opt1, gc).Column D of Table 1 provides SEQ ID NO of the corresponding G/C contentmodified nucleic acid coding sequences (opt10, gc mod) (for a detaileddescription of “coding sequences”, see paragraph “suitable codingsequences”).

Notably, the description of the invention explicitly includes theinformation provided under <223> identifier of the ST25 sequence listingof the present application. Preferred RNA constructs comprising codingsequences of Table 1, e.g. mRNA sequences comprising the codingsequences of Table 1, are provided in Table 2.

TABLE 1 Preferred SARS-CoV-2 constructs (amino acid sequences andnucleic acid coding sequences): row A B C D 1 Full-length spike protein;S - WT   1  136 2 Stabilized spike proteins; S_stab_PP; the RNA sequenceencoding S- 10, 137, 146, stab_PP but with wt codon usage is provided bySEQ ID NO: 28916 22738, 22765, 23150- 22740, 22767, 23184, 22742, 22769,27202- 22744, 22771, 27247 22746, 22773, 22748, 22775, 22750, 22777,22752, 22779, 22754, 22781, 22756, 22783, 22758, 22785, 22959- 23089-22964, 23148, 27087- 27110- 27109, 27201, 28540- 28589- 28588 28637 3Spike protein receptor binding domain; RBD 13243, 22917, 22923, 27007-27046 4 Spike protein critical neutralisation domain; CND 13310, 27047-27086 5 A222V_D614G 22740 22767 23151 S_stab_PP(K986P_V987P_A222V_D614G)6 N439K_D614G 22742 22769 23152 S_stab_PP(K986P_V987P_N439K_D614G) 7S477N_D614G 22744 22771 23153 S_stab_PP(K986P_V987P_S477N_D614G) 8N501Y_D614G 22746 22773 23154 S_stab_PP(K986P_V987P_N501Y_D614G) 9H69del_V70del_D614G 22748 22775 23155S_stab_PP(K986P_V987P_H69del_V70del_D614G) 10 Y453F_D614G 22750 2277723156 S_stab_PP(K986P_V987P_Y453F_D614G) 11 I692V_D614G 22752 2277923157 S_stab_PP(K986P_V987P_D614G_I692V) 12 M1229I_D614G 22754 2278123158 S_stab_PP(K986P_V987P_D614G_M1229I) 13 MINK1 (w/oM1229I) 2275622783 23159S_stab_PP(K986P_V987P_H69del_V70del_A222V_Y453F_S477N_D614G_I692V) 14MINK2 22758 22785 23160S_stab_PP(K986P_V987P_H69del_V70del_Y453F_D614G_I692V_M1229I) 15 B.1.1.7(UK) 22959 23089 23161S_stab_PP(K986P_V987P_H69del_V70del_Y144del_N501Y_A570D_D614G_(—)P681H_T716I_S982A_D1118H) 16 minSA (B.1.351) 22960 23090 23162S_stab_PP(K986P_V987P_K417N_E484K_N501Y_D614G) 17 fullSA (B.1.351) 2296123091 23163S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_A243del_L244del_(—)R246I_K417N_E484K_N501Y_D614G_A701V) 18 E484K 22962 23092 23164S_stab_PP(K986P_V987P_E484K_D614G) 19 P.1 (Brazil) 22963 23093 23165S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_K417T_E484K_(—)N501Y_D614G_H655Y_T1027I) 20 B.1.429 (California) 22964 23094 23166S_stab_PP(K986P_V987P_S13I_W152C_L452R_D614G) 21 B.1.1.7 + E484K 2708727110 27202S_stab_PP(K986P_V987P_H69del_V70del_Y144del_E484K_N501Y_A570D_(—)D614G_P681H_T716I_S982A_D1118H) 22 fullSAminusI246R 27088 27111 27203S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_A243del_L244del_(—)K417N_E484K_N501Y_D614G_A701V) 23 B.1.525 (Nigeria) 27089 27112 27204S_stab_PP(K986P_V987P_Q52R_A67V_H69del_V70del_Y144del_E484K_(—)D614G_Q677H_F888L) 24 B.1.525_v2 (Nigeria) 27090 27113 27205S_stab_PP(K986P_V987P_A67V_H69del_V70del_Y144del_E484K_D614G_(—)Q677H_F888L) 25 P.1 + V1176F (Brazil) 27091 27114 27206S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_K417T_(—)E484K_N501Y_D614G_H655Y_T1027I_V1176F) 26 P.2 (Brazil) 27092 27115 27207S_stab_PP(K986P_V987P_E484K_D614G_V1176F) 27 B.1.617 (India) 27093 2711627208 S_stab_PP(K986P_V987P_L452R_D614G_P681R) 28 B.1.617.1 (India)27094 27117 27209S_stab_PP(K986P_V987P_E154K_L452R_E484Q_D614G_P681R_Q1071H) 29 B.1.617.2(India) 27095 27118 27210S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_T478K_D614G_(—)P681R_D950N) 30 C.37.1 (Peru) 27096 27119 27211S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_Y248del_L249del_(—)T250del_P251del_G252del_L452Q_F490S_D614G_T859N) 31 B.1.258(CzechRepublic) 27097 27120 27212S_stab_PP(K986P_V987P_H69del_V70del_N439K_D614G) 32 B.1.526 (NewYork)27098 27121 27213S_stab_PP(K986P_V987P_L5F_T95I_D253G_E484K_D614G_A701V) 33 B.1.526.2(NewYork) 27099 27122 27214S_stab_PP(K986P_V987P_L5F_T95I_D253G_S477N_D614G_Q957R) 34 A.23.1_v1(Rwanda/Uganda) 27100 27123 27215S_stab_PP(K986P_V987P_F157L_V367F_Q613H_P681R) 35 A.23.1_v2(Rwanda/Uganda) 27101 27124 27216S_stab_PP(K986P_V987P_S254F_D614G_P681R_G769V) 36 B.1.620(unclear/Africa) 27102 27125 27217S_stab_PP(K986P_V987P_P26S_H69del_V70del_V126A_Y144del_L242del_(—)A243del_L244del_H245Y_S477N_E484K_D614G_P681H_T1027I_D1118H) 37 B.1.621(Columbia) 27103 27126 27218S_stab_PP(K986P_V987P_T95I_Y144T_Y145S_ins145N_R346K_E484K_(—)N501Y_D614G_P681H_D950N) 38 B.1.214.2 (unclear) 27104 27127 27219S_stab_PP(K986P_V987P_ins214TDR_Q414K_N450K_D614G_T716I) 39 B.1.1.519(Mexico) 27105 27128 27220S_stab_PP(K986P_V987P_T478K_D614G_P681H_T732A) 40 P.3 (Philippines)27106 27129 27221S_stab_PP(K986P_V987P_E484K_N501Y_D614G_P681H_E1092K_H1101Y_V1176F) 41B.1.616 (France) 27107 27130 27222S_stab_PP(K986P_V987P_H66D_G142V_Y144del_Y145del_D215G_V483A_(—)D614G_H655Y_G669S_Q949R_N1187D) 42 v1 (Vietnam) 27108 27131 27223S_stab_PP(K986P_V987P_Y144del_L452R_T478K_P681R) 43 v2 (Vietnam) 2710927132 27224S_stab_PP(K986P_V987P_T19R_Y144del_Y145del_L452R_T478K_D614G_P681R) 44C.1.2 (SouthAfrica2) 28540 28589S_stab_PP(K986P_V987P_P9L_C136F_Y144del_R190S_D215G_L242del_(—)A243del_Y449H_E484K_N501Y_D614G_H655Y_N679K_T716I_T859N) 45 BA.1_v1(SouthAfrica3) 28541 28590S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_(—)D614G_H655Y_N679K_P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v1) 46 S_stab_PP(K986P BA.1_v0 (SouthAfrica3)28542 28591S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)K417N_N440K_G446S_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_(—)T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v0) 47 B.1.1.529 (SouthAfrica3) 28543 28592S_stab_PP(K986P_V987P_A67V_T95I_G339D_S371L_S373P_S375F_S477N_(—)T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_H655Y_(—)N679K_P681H_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_B.1.1.529) 48 BA.2 (SouthAfrica3) 28544 28593S_stab_PP(K986P_V987P_T19I_L24del_P25del_P26del_A27S_G142D_V213G_(—)G339D_S371F_S373P_S375F_T376A_D405N_S477N_T478K_E484A_Q493R_(—)Q498R_N501Y_Y505H_D614G_H655Y_N679K_P681H_D796Y_Q954H_N969K);S_stab_PP(K986P_V987P_BA.2) 49 C.37_v2 (Peru/Lima) 28545 28594S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_Y248del_L249del_(—)T250del_P251del_G252del_D253N_L452Q_F490S_D614G_T859N) 50 B.1.621_v2(Columbia) 28546 28595S_stab_PP(K986P_V987P_T95I_Y144S_Y145N_R346K_E484K_N501Y_D614G_(—)P681H_D950N) 51 C.36.3 (Thailand) 28547 28596S_stab_PP(K986P_V987P_S12F_H69del_V70del_W152R_R346S_L452R_(—)D614G_Q677H_A899S) 52 B.1.619 (Cameroon) 28548 28597S_stab_PP(K986P_V987P_I210T_N440K_E484K_D614G_D936N_S939F_T1027I) 53 R.1(Kentucky) 28549 28598 S_stab_PP(K986P_V987P_W152L_E484K_D614G_G769V) 54B.1.1.176 (Canada) 28550 28599 S_stab_PP(K986P_V987P_T20I_R357K_D614G)55 AZ.3 28551 28600S_stab_PP(K986P_V987P_T95I_Y144del_E484K_D614G_P681H_D796H) 56B.1.617.2_v2 (India) 28552 28601S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_L452R_(—)T478K_D614G_P681R_D950N) 57 AY.1 (India) 28553 28602S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)W258L_K417N_L452R_T478K_D614G_P681R_D950N) 58 AY.2 (India) 28554 28603S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_R158del_(—)A222V_K417N_L452R_T478K_D614G_P681R_D950N) 59 AY.4_v1 (India) 2855528604 S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)L452R_T478K_D614G_P681R_D950N) 60 AY.4_v2 (India) 28556 28605S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_L452R_T478K_(—)D614G_P681R_D950N) 61 AY.4.2 (India) 28557 28606S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_F157del_(—)R158del_A222V_L452R_T478K_D614G_P681R_D950N) 62 B.1.617.3 (India) 2855828607 S_stab_PP(K986P_V987P_T19R_L452R_E484Q_D614G_P681R_D950N) 63B.1.617 (India)_w/oFCS 28559 28608S_stab_PP(K986P_V987P_L452R_D614G_P681R_R682del_R683del_A684del_R685del)64 B.1.617.1 (India)_w/oFCS 28560 28609S_stab_PP(K986P_V987P_E154K_L452R_E484Q_D614G_P681R_R682del_(—)R683del_A684del_R685del_Q1071H) 65 B.1.617.2 (India)_w/oFCS 28561 28610S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_T478K_D614G_(—)P681R_R682del_R683del_A684del_R685del_D950N) 66 B.1,617.2_v2(India)_w/oFCS 28562 28611S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_L452R_(—)T478K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 67 AY.1(India)_w/oFCS 28563 28612S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_W258L_(—)K417N_L452R_T478K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 68AY.2 (India)_w/oFCS 28564 28613S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_R158del_A222V_(—)K417N_L452R_T478K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 69AY.4_v1 (India)_w/oFCS 28565 28614S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)L452R_T478K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 70AY.4_v2 (India)_w/oFCS 28566 28615S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_L452R_T478K_D614G_(—)P681R_R682del_R683del_A684del_R685del_D950N) 71 AY.4.2 (India)_w/oFCS28567 28616S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_F157del_R158del_(—)A222V_L452R_T478K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 72B.1.617.3 (India)_w/oFCS 28568 28617S_stab_PP(K986P_V987P_T19R_L452R_E484Q_D614G_P681R_R682del_(—)R683del_A684del_R685del_D950N) 73 B.1.617 (India)_withE484K 28569 28618S_stab_PP(K986P_V987P_L452R_E484K_D614G_P681R) 74 B.1.617.1(India)_withE484K 28570 28619S_stab_PP(K986P_V987P_E154K_L452R_E484K_D614G_P681R_Q1071H) 75 B.1.617.2(India)_withE484K 28571 28620S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_T478K_E484K_(—)D614G_P681R_D950N) 76 B.1.617.2_v2 (India)_withE484K 28572 28621S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_L452R_(—)T478K_E484K_D614G_P681R_D950N) 77 AY.1 (India)_withE484K 28573 28622S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)W258L_K417N_L452R_T478K_E484K_D614G_P681R_D950N) 78 AY.2(India)_withE484K 28574 28623S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_R158del_(—)A222V_K417N_L452R_T478K_E484K_D614G_P681R_D950N) 79 AY.4_v1(India)_withE484K 28575 28624S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)L452R_T478K_E484K_D614G_P681R_D950N) 80 AY.4_v2 (India)_withE484K 2857628625 S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_L452R_T478K_(—)E484K_D614G_P681R_D950N) 81 AY.4.2 (India)_withE484K 28577 28626S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_F157del_(—)R158del_A222V_L452R_T478K_E484K_D614G_P681R_D950N) 82 B.1.617.3(India)_withE484K 28578 28627S_stab_PP(K986P_V987P_T19R_L452R_E484K_D614G_P681R_D950N) 83 B.1.617(India)_withE484K_w/oFCS 28579 28628S_stab_PP(K986P_V987P_L452R_E484K_D614G_P681R_R682del_R683del_(—)A684del_R685del) 84 B.1.617.1 (India)_withE484K_w/oFCS 28580 28629S_stab_PP(K986P_V987P_E154K_L452R_E484K_D614G_P681R_R682del_(—)R683del_A684del_R685del_Q1071H) 85 B.1.617.2 (India)_withE484K_w/oFCS28581 28630S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_T478K_E484K_(—)D614G_P681R_R682del_R683del_A684del_R685del_D950N) 86 B.1.617.2_v2(India)_withE484K_w/oFCS 28582 28631S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_L452R_(—)T478K_E484K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 87 AY.1(India)_withE484K_w/oFCS 28583 28632S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_(—)W258L_K417N_L452R_T478K_E484K_D614G_P681R_R682del_R683del_(—)A684del_R685del_D950N) 88 AY.2 (India)_withE484K_w/oFCS 28584 28633S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_R158del_(—)A222V_K417N_L452R_T478K_E484K_D614G_P681R_R682del_R683del_(—)A684del_R685del_D950N) 89 AY.4_v1 (India)_withE484K_w/oFCS 28585 28634S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_R158del_L452R_(—)T478K_E484K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 90AY.4_v2 (India)_withE484K_w/oFCS 28586 28635S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_L452R_T478K_(—)E484K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 91 AY.4.2(India)_withE484K_w/oFCS 28587 28636S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_F157del_(—)R158del_A222V_L452R_T478K_E484K_D614G_P681R_R682del_R683del_(—)A684del_R685del_D950N) 92 B.1.617.3 (India)_withE484K_w/oFCS 28588 28637S_stab_PP(K986P_V987P_T19R_L452R_E484K_D614G_P681R_R682del_(—)R683del_A684del_R685del_D950N) 93 BA.1_v2 (SouthAfrica3) 28917 28921S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)N440K_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_(—)H655Y_N679K_P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v2) 94 BA.1_v3 (SouthAfrica3) 28918 28922S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_(—)S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_(—)D614G_H655Y_N679K_P681H_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v3) 95 BA.1_v4 (SouthAfrica3) 28919 28923S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_(—)H655Y_N679K_P681H_A701V_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v4) 96 BA.1_v5 (SouthAfrica3) 28920 28924S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_V143del_(—)Y144del_Y145del_N211del_L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)G446S_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_T547K_(—)D614G_H655Y_N679K_P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v5) w/oFCS: deleted furin cleavage site

Suitable Coding Sequences:

According to preferred embodiments, the RNA of the invention comprisesat least one coding sequence encoding at least one antigenic peptide orprotein selected from or derived from a SARS-CoV-2 spike protein,preferably as defined above, or fragments and variants thereof. In thatcontext, any coding sequence encoding at least one antigenic proteinSARS-CoV-2 spike protein as defined herein, or fragments and variantsthereof may be understood as suitable coding sequence and may thereforebe comprised in the RNA of the invention.

In preferred embodiments , the RNA of the first aspect may comprise orconsist of at least one coding sequence encoding at least one antigenicpeptide or protein from SARS-CoV-2 as defined herein, preferablyencoding any one of SEQ ID NOs: 1, 10, 22738, 22740, 22742, 22744,22746, 22748, 22750, 22752, 22754, 22756, 22758, 22959-22964,27087-27109, 28540-28588, 28917-28920 or fragments or variants thereof.It has to be understood that, on nucleic acid level, any RNA sequencewhich encodes an amino acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 116, 136, 137, 146,22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783,22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916,28921-28924 or fragments or variants thereof, may be selected and mayaccordingly be understood as suitable coding sequence of the invention.In certain embodiments the RNA sequence which encodes a SARS-CoV-2 spikeprotein is at least 95% identical to any one of SEQ ID NOs: 116, 136,137, 146, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781,22783, 22785, 23089-23148, 23150-23184, 27110-27247, 28589-28637, 28916,or 28921-28924.

In preferred embodiments, the RNA of the first aspect may comprise orconsist of at least one coding sequence encoding at least one antigenicpeptide or protein from SARS-CoV-2 as defined herein, preferablyencoding any one of SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746,22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109,28540-28588, 28917-28920 or fragments or variants thereof. It has to beunderstood that, on nucleic acid level, any RNA sequence which encodesan amino acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 137, 146, 22765, 22767, 22769,22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148,23150-23184, 23095-23112, 27110-27247, 28589-28637, 28921-28924 orfragments or variants thereof, may be selected and may accordingly beunderstood as suitable coding sequence of the invention. In certainembodiments the RNA sequence which encodes a SARS-CoV-2 spike protein isat least 95% identical to any one of SEQ ID NOs: 137, 146, 22765, 22767,22769, 22771, 22773, 22775, 22777, 22779, 22781, 22783, 22785,23089-23148, 23150-23184, 27110-27247 28589-28637, or 28921-28924.

In preferred embodiments, the RNA of the first aspect may comprise orconsist of at least one coding sequence encoding at least one antigenicpeptide or protein from SARS-CoV-2 as defined herein, preferablyencoding any one of SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746,22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109,28540-28588, 28917-28920 or fragments or variants thereof. It has to beunderstood that, on nucleic acid level, any RNA sequence which encodesan amino acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 137, 22765, 22767, 22769, 22771,22773, 22775, 22777, 22779, 22781, 22783, 22785, 23089-23148,27110-27201, 28589-28637, 28921-28924 or fragments or variants thereof,may be selected and may accordingly be understood as suitable codingsequence of the invention. In certain embodiments the RNA sequence whichencodes a SARS-CoV-2 spike protein is at least 95% identical to any oneof SEQ ID NOs: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777,22779, 22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, or28921-28924.

In preferred embodiments, the RNA of the first aspect may comprise orconsist of at least one coding sequence encoding at least one antigenicpeptide or protein from SARS-CoV-2 as defined herein, preferablyencoding any one of SEQ ID NOs: 10, 22738, 22740, 22742, 22744, 22746,22748, 22750, 22752, 22754, 22756, 22758, 22959-22964, 27087-27109,28540-28588, 28917-28920 or fragments or variants thereof. It has to beunderstood that, on nucleic acid level, any RNA sequence which encodesan amino acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 146, 23150-23184, 27202-27247 orfragments or variants thereof, may be selected and may accordingly beunderstood as suitable coding sequence of the invention. In certainembodiments the RNA sequence which encodes a SARS-CoV-2 spike protein isat least 95% identical to any one of SEQ ID NOs: 146, 23150-23184 or27202-27247.

In preferred embodiments, the RNA of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording to SEQ ID NOs: 22765, 22767, 22769, 22771, 22773, 22775,22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184,27110-27247, 28589-28637, 28921-28924 or a fragment or a fragment orvariant of any of these sequences. In certain embodiments the RNAsequence which encodes a SARS-CoV-2 spike protein is at least 95%identical to any one of SEQ ID NOs: 22765, 22767, 22769, 22771, 22773,22775, 22777, 22779, 22781, 22783, 22785, 23089-23148, 23150-23184,27110-27247, 28589-28637, or 28921-28924. Additional informationregarding each of these suitable nucleic acid sequences may also bederived from the sequence listing, in particular from the detailsprovided therein under identifier <223>.

In preferred embodiments, the RNA of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording to SEQ ID NOs: 22765, 22767, 22769, 22771, 22773, 22775,22777, 22779, 22781, 22783, 22785, 23089-23094, 27110-27132,28589-28637, or 28921-28924 or a fragment or a fragment or variant ofany of these sequences. In certain embodiments the RNA sequence whichencodes a SARS-CoV-2 spike protein is at least 95% identical to any oneof SEQ ID NOs: 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779,22781, 22783, 22785, 23089-23094, 27110-27132, 28589-28637, or28921-28924.

In preferred embodiments, the RNA of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording to SEQ ID NOs: 23150-23166 or 27202-27224 or a fragment or afragment or variant of any of these sequences. In certain embodimentsthe RNA sequence which encodes a SARS-CoV-2 spike protein is at least95% identical to any one of SEQ ID NOs: 23150-23166 or 27202-27224.

In preferred embodiments, the RNA of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording to SEQ ID NOs: 23150-23166, 27202-27224, 23114-23130 or27156-27178 or a fragment or a fragment or variant of any of thesesequences. In certain embodiments the RNA sequence which encodes aSARS-CoV-2 spike protein is at least 95% identical to any one of SEQ IDNOs: 23114-23130 or 27156-27178.

In preferred embodiments, the RNA of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording to SEQ ID NOs: 23150-23166, 27202-27224, 23167-23184 or27225-27247 or a fragment or a fragment or variant of any of thesesequences. In certain embodiments the RNA sequence which encodes aSARS-CoV-2 spike protein is at least 95% identical to any one of SEQ IDNOs: 23167-23184 or 27225-27247.

In preferred embodiments, the RNA of the first aspect is an artificialRNA.

The term “artificial RNA” as used herein is intended to refer to an RNAthat does not occur naturally. In other words, an artificial RNA may beunderstood as a non-natural RNA molecule. Such RNA molecules may benon-natural due to its individual sequence (e.g. G/C content modifiedcoding sequence, UTRs) and/or due to other modifications, e.g.structural modifications of nucleotides. Typically, artificial RNA maybe designed and/or generated by genetic engineering to correspond to adesired artificial sequence of nucleotides. In this context, anartificial RNA is a sequence that may not occur naturally, i.e. asequence that differs from the wild type sequence/the naturallyoccurring sequence by at least one nucleotide. The term “artificial RNA”is not restricted to mean “one single RNA molecule” but is understood tocomprise an ensemble of essentially identical RNA molecules.Accordingly, it may relate to a plurality of essentially identical RNAmolecules.

In preferred embodiments, the RNA of the first aspect is a modifiedand/or stabilized RNA, preferably a modified and/or stabilizedartificial RNA.

According to preferred embodiments, the RNA of the present invention maythus be provided as a “stabilized artificial RNA” or “stabilized codingRNA” that is to say an RNA showing improved resistance to in vivodegradation and/or an RNA showing improved stability in vivo, and/or anRNA showing improved translatability in vivo. In the following, specificsuitable modifications/adaptations in this context are described whichare suitably to “stabilize” the RNA. Preferably, the RNA of the presentinvention may be provided as a “stabilized RNA” or “stabilized codingRNA”.

Such stabilization may be affected by providing a “dried RNA” and/or a“purified RNA” as further specified below. Alternatively, or in additionto that, such stabilization can be affected, for example, by a modifiedphosphate backbone of the RNA of the present invention. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in thenucleic acid are chemically modified. Nucleotides that may be used inthis connection contain e.g. a phosphorothioate-modified phosphatebackbone, preferably at least one of the phosphate oxygens contained inthe phosphate backbone being replaced by a sulfur atom. Stabilized RNAsmay further include, for example: non-ionic phosphate analogues, suchas, for example, alkyl and aryl phosphonates, in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, suitable modifications are described that are capableof “stabilizing” the RNA of the invention.

In preferred embodiments, the RNA comprises at least one codon modifiedcoding sequence.

In preferred embodiments, the at least one coding sequence of the RNA isa codon modified coding sequence, wherein the amino acid sequenceencoded by the at least one codon modified coding sequence is preferablynot being modified compared to the amino acid sequence encoded by thecorresponding wild type coding sequence or reference coding sequence.

The term “codon modified coding sequence” relates to coding sequencesthat differ in at least one codon (triplets of nucleotides coding forone amino acid) compared to the corresponding wild type or referencecoding sequence. Suitably, a codon modified coding sequence in thecontext of the invention may show improved resistance to in vivodegradation and/or improved stability in vivo, and/or improvedtranslatability in vivo. Codon modifications in the broadest sense makeuse of the degeneracy of the genetic code wherein multiple codons mayencode the same amino acid and may be used interchangeably tooptimize/modify the coding sequence for in vivo applications.

The term “reference coding sequence” relates to the coding sequence,which was the origin sequence to be modified and/or optimized.

In preferred embodiments, the at least one coding sequence of the RNA isa codon modified coding sequence, wherein the codon modified codingsequence is selected from C maximized coding sequence, CAI maximizedcoding sequence, human codon usage adapted coding sequence, G/C contentmodified coding sequence, and G/C optimized coding sequence, or anycombination thereof.

In preferred embodiments, the at least one coding sequence of the RNAhas a G/C content of at least about 50%, 55%, or 60%. In particularembodiments, the at least one coding sequence of the RNA of component Ahas a G/C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.

When transfected into mammalian host cells, the RNA comprising a codonmodified coding sequence has a stability of between 12-18 hours, orgreater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72hours and are capable of being expressed by the mammalian host cell(e.g. a muscle cell).

When transfected into mammalian host cells, the RNA comprising a codonmodified coding sequence is translated into protein, wherein the amountof protein is at least comparable to, or preferably at least 10% morethan, or at least 20% more than, or at least 30% more than, or at least40% more than, or at least 50% more than, or at least 100% more than, orat least 200% or more than the amount of protein obtained by a naturallyoccurring or wild type or reference coding sequence transfected intomammalian host cells.

In some embodiments, the RNA may be modified, wherein the C content ofthe at least one coding sequence may be increased, preferably maximized,compared to the C content of the corresponding wild type or referencecoding sequence (herein referred to as “C maximized coding sequence”).The generation of a C maximized nucleic acid sequences may suitably becarried out using a modification method according to WO2015/062738. Inthis context, the disclosure of WO2015/062738 is included herewith byreference.

In preferred embodiments, the RNA may be modified, wherein the G/Ccontent of the at least one coding sequence may be optimized compared tothe G/C content of the corresponding wild type or reference codingsequence (herein referred to as “G/C content optimized codingsequence”). “Optimized” in that context refers to a coding sequencewherein the G/C content is preferably increased to the essentiallyhighest possible G/C content. The generation of a G/C content optimizedRNA sequences may be carried out using a method according toWO2002/098443. In this context, the disclosure of WO2002/098443 isincluded in its full scope in the present invention. Throughout thedescription, including the <223> identifier of the sequence listing, G/Coptimized coding sequences are indicated by the abbreviations “opt1” or“gc”.

In preferred embodiments, the RNA may be modified, wherein the codons inthe at least one coding sequence may be adapted to human codon usage(herein referred to as “human codon usage adapted coding sequence”).Codons encoding the same amino acid occur at different frequencies inhumans. Accordingly, the coding sequence of the nucleic acid ispreferably modified such that the frequency of the codons encoding thesame amino acid corresponds to the naturally occurring frequency of thatcodon according to the human codon usage. For example, in the case ofthe amino acid Ala, the wild type or reference coding sequence ispreferably adapted in a way that the codon “GCC” is used with afrequency of 0.40, the codon “GCT” is used with a frequency of 0.28, thecodon “GCA” is used with a frequency of 0.22 and the codon “GCG” is usedwith a frequency of 0.10 etc. Accordingly, such a procedure (asexemplified for Ala) is applied for each amino acid encoded by thecoding sequence of the nucleic acid to obtain sequences adapted to humancodon usage. Throughout the description, including the <223> identifierof the sequence listing, human codon usage adapted coding sequences areindicated by the abbreviation “opt3” or “human”.

In some embodiments, the RNA may be modified, wherein the G/C content ofthe at least one coding sequence may be modified compared to the G/Ccontent of the corresponding wild type or reference coding sequence(herein referred to as “G/C content modified coding sequence”). In thiscontext, the terms “G/C optimization” or “G/C content modification”relate to a nucleic acid that comprises a modified, preferably anincreased number of guanosine and/or cytosine nucleotides as compared tothe corresponding wild type or reference coding sequence. Such anincreased number may be generated by substitution of codons containingadenosine or thymidine nucleotides by codons containing guanosine orcytosine nucleotides. Advantageously, nucleic acid sequences having anincreased G/C content are more stable or show a better expression thansequences having an increased A/U. Preferably, the G/C content of thecoding sequence of the nucleic acid is increased by at least 10%, 20%,30%, preferably by at least 40% compared to the G/C content of thecoding sequence of the corresponding wild type or reference nucleic acidsequence (herein referred to “opt 10” or “gc mod”). For example, the theG/C content of the coding sequence of the nucleic acid is preferablyincreased by at least 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24% or 25% relative to the G/C content of SEQ ID NO: 28916.

In some embodiments, the RNA may be modified, wherein the codonadaptation index (CAI) may be increased or preferably maximised in theat least one coding sequence (herein referred to as “CAI maximizedcoding sequence”). It is preferred that all codons of the wild type orreference nucleic acid sequence that are relatively rare in e.g. a humanare exchanged for a respective codon that is frequent in the e.g. ahuman, wherein the frequent codon encodes the same amino acid as therelatively rare codon. Suitably, the most frequent codons are used foreach amino acid of the encoded protein. Suitably, the RNA comprises atleast one coding sequence, wherein the codon adaptation index (CAI) ofthe at least one coding sequence is at least 0.5, at least 0.8, at least0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI)of the at least one coding sequence is 1 (CAI=1). For example, in thecase of the amino acid Ala, the wild type or reference coding sequencemay be adapted in a way that the most frequent human codon “GCC” isalways used for said amino acid. Accordingly, such a procedure (asexemplified for Ala) may be applied for each amino acid encoded by thecoding sequence of the nucleic acid to obtain CAI maximized codingsequences.

In particularly preferred embodiments, the at least one coding sequenceof the nucleic acid is a codon modified coding sequence, wherein thecodon modified coding sequence is a G/C optimized coding sequence.

In particularly preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a G/Coptimized nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 137, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779,22781, 22783, 22785, 23089-23148, 27110-27201, 28589-28637, 28921-28924or a fragment or variant of any of these sequences.

In particularly preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a G/Coptimized nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 146, 23150-23184, 27202-27247 or a fragment or variant ofany of these sequences.

In even more preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codonmodified nucleic acid sequence selected from the group consisting of SEQID NOs: 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109, 23127,23145, 23163, 23181, 28589 (B.1.315; C.1.2) or a fragment or variant ofany of these sequences. In some aspects, the at least one codingsequence encoding the SARS-CoV-2 antigen is at least 95% identical toSEQ ID NOs: 23090, 23108, 23126, 23144, 23162, 23180, 23091, 23109,23127, 23145, 23163, 23181 or 28589. In some aspects, the at least onecoding sequence encoding the SARS-CoV-2 antigen is at least 95%identical to SEQ ID NOs: 23090-23091, 23162-23163 or 28589.

In even more preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codonmodified nucleic acid sequence selected from the group consisting of SEQID NOs: 27116, 27139, 27162, 27185, 27208, 27231, 27117, 27140, 27163,27186, 27209, 27232, 27118, 27141, 27164, 27187, 27210, 27233,28601-28607 (B.1.617; B.1.617.1; B.1.617.2; AY.1; AY.2; AY.4; AY.4.2;B.1.617.3) or a fragment or variant of any of these sequences. In someaspects, the at least one coding sequence encoding the SARS-CoV-2antigen is at least 95% identical to SEQ ID NOs: 27116, 27139, 27162,27185, 27208, 27231, 27117, 27140, 27163, 27186, 27209, 27232, 27118,27141, 27164, 27187, 27210, 27233, or 28601-28607. In some aspects, theat least one coding sequence encoding the SARS-CoV-2 antigen is at least95% identical to SEQ ID NOs: 27116-27118 27208-27210 or 28601-28607.

In even more preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codonmodified nucleic acid sequence selected from the group consisting of SEQID NOs: 27118, 27141, 27164, 27187, 27210, 27233 or 28601-28606(B.1.617.2; AY.1; AY.2; AY.4; AY.4.2) or a fragment or variant of any ofthese sequences. In some aspects, the at least one coding sequenceencoding the SARS-CoV-2 antigen is at least 95% identical to SEQ ID NOs:27118, 27141, 27164, 27187, 27210, 27233 or 28601-28606. In someaspects, the at least one coding sequence encoding the SARS-CoV-2antigen is at least 95% identical to SEQ ID NOs: 27118, 27210 or28601-28606.

In even more preferred embodiments, the RNA of the first aspectcomprises at least one coding sequence comprising or consisting a G/Coptimized coding sequence encoding the SARS-CoV-2 antigen as definedherein which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codonmodified nucleic acid sequence selected from the group consisting of SEQID NOs: 27118, 27141, 27164, 27187, 27210 or 27233 (B.1.617.2) or afragment or variant of any of these sequences. In some aspects, the atleast one coding sequence encoding the SARS-CoV-2 antigen is at least95% identical to SEQ ID NOs: 27118, 27141, 27164, 27187, 27210 or 27233.In some aspects, the at least one coding sequence encoding theSARS-CoV-2 antigen is at least 95% identical to SEQ ID NOs: 27118 or27210.

In further preferred embodiments, the RNA of the first aspect comprisesat least one coding sequence comprising or consisting a G/C optimizedcoding sequence encoding the SARS-CoV-2 antigen as defined herein whichis identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codon modifiednucleic acid sequence selected from the group consisting of SEQ ID NOs:28590-28593, 28921-28924 (B.1.1.529, Omicron) or a fragment or variantof any of these sequences. In some aspects, the at least one codingsequence encoding the SARS-CoV-2 antigen is at least 95% identical toSEQ ID NOs: 28590-28593. In some aspects, the at least one codingsequence encoding the SARS-CoV-2 antigen is at least 95% identical toSEQ ID NOs: 28590-28593, 28921-28924.

UTRs:

In preferred embodiments, the RNA of the invention comprises at leastone coding sequence encoding at least one SARS-CoV-2 spike protein asdefined herein, or an immunogenic fragment or immunogenic variantthereof, wherein the RNA comprises at least one heterologousuntranslated region (UTR). In some aspects a RNA of the embodiments doesnot comprise a 3′ UTR comprising the sequence of SEQ ID NO: 268. Incertain aspects, a RNA of the embodiments comprises a 3′ UTR comprisingthe sequence of SEQ ID NO: 268

In preferred embodiments, the RNA of the invention comprises aprotein-coding region (“coding sequence” or “cds”), and 5′-UTR and/or3′-UTR. Notably, UTRs may harbor regulatory sequence elements thatdetermine nucleic acid, e.g. RNA turnover, stability, and localization.Moreover, UTRs may harbor sequence elements that enhance translation. Inmedical applications, translation of the RNA into at least one peptideor protein is of paramount importance to therapeutic efficacy. Certaincombinations of 3′-UTRs and/or 5′-UTRs may enhance the expression ofoperably linked coding sequences encoding peptides or proteins of theinvention. RNA molecules harboring said UTR combinations advantageouslyenable rapid and transient expression of antigenic peptides or proteinsafter administration to a subject, preferably after intramuscularadministration. Accordingly, the RNA comprising certain combinations of3′-UTRs and/or 5′-UTRs as provided herein is particularly suitable foradministration as a vaccine, in particular, suitable for administrationinto the muscle, the dermis, or the epidermis of a subject.

Suitably, the RNA of the invention comprises at least one heterologous5′-UTR and/or at least one heterologous 3′-UTR. Said heterologous5′-UTRs or 3′-UTRs may be derived from naturally occurring genes or maybe synthetically engineered. In preferred embodiments, the RNA comprisesat least one coding sequence as defined herein operably linked to atleast one (heterologous) 3′-UTR and/or at least one (heterologous)5′-UTR.

In preferred embodiments, the RNA comprises at least one heterologous3′-UTR, wherein the RNA does not comprise a 3′-UTR comprising thesequence of SEQ ID NO: 268. Preferably, the RNA comprises a 3′-UTR,which may be derivable from a gene that relates to an RNA with enhancedhalf-life (i.e. that provides a stable RNA).

In some embodiments, a 3′-UTR comprises one or more of a polyadenylationsignal, a binding site for proteins that affect a nucleic acid stabilityor location in a cell, or one or more miRNA or binding sites for miRNAs.

MicroRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′-UTR of nucleic acid molecules and down-regulate geneexpression either by reducing nucleic acid molecule stability or byinhibiting translation. E.g., microRNAs are known to regulate RNA, andthereby protein expression, e.g. in liver (miR-122), heart (miR-Id,miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7,miR-30c), kidney (miR-192, miR-194, miR-204), myeloid cells (miR-142-3p,miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133,miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126).The RNA may comprise one or more microRNA target sequences, microRNAsequences, or microRNA seeds. Such sequences may e.g. correspond to anyknown microRNA such as those taught in US2005/0261218 andUS2005/0059005.

Accordingly, miRNA, or binding sites for miRNAs as defined above may beremoved from the 3′-UTR or introduced into the 3′-UTR in order to tailorthe expression of the RNA to desired cell types or tissues (e.g. musclecells).

In preferred embodiments, the RNA comprises at least one heterologous3′-UTR that comprises or consists of a nucleic acid sequence derivedfrom a 3′-UTR of a gene selected from PSMB3, ALB7, CASP1, COX6B1, GNAS,NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any oneof these genes. In some embodiments, the RNA comprises at least oneheterologous 3′-UTR, wherein the at least one heterologous 3′-UTRcomprises a nucleic acid sequence derived from a 3′-UTR of a geneselected from PSMB3, ALB7, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or froma homolog, a fragment or variant of any one of these genes, preferablyaccording to nucleic acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 253-266, 22902-22905, 22876-22895,26996-26999, 28528-28539 or a fragment or a variant of any of these.Particularly preferred nucleic acid sequences in that context can bederived from published PCT application WO2019/077001A1, in particular,claim 9 of WO2019/077001A1. The corresponding 3′-UTR sequences of claim9 of WO2019/077001A1 are herewith incorporated by reference (e.g., SEQID NOs: 23-34 of WO2019/077001A1, or fragments or variants thereof).

In further embodiments, the RNA comprises a 3′-UTR derived from a RPS9gene. Said 3′-UTR derived from a RPS9 gene may comprise or consist of anucleic acid sequence being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NOs: 263 or 264, 22894, 22895, 22904, 22905 or afragment or a variant thereof.

In preferred embodiments, the RNA comprises a 3′-UTR derived from aPSMB3 gene. Said 3′-UTR derived from a PSMB3 gene may comprise orconsist of a nucleic acid sequence being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NOs: 253 or 254, 22892, 22893, 22902, 22903,26996-26999, 28528-28539 or a fragment or a variant thereof.

In other embodiments, the RNA comprises a 3′-UTR which comprises orconsists of a nucleic acid sequence being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 22876-22891, 28526, 28527 or afragment or a variant thereof.

In other embodiments, the RNA may comprise a 3′-UTR as described inWO2016/107877, the disclosure of WO2016/107877 relating to 3′-UTRsequences herewith incorporated by reference. Suitable 3′-UTRs are SEQID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016/107877, or fragments orvariants of these sequences. In other embodiments, the nucleic acidcomprises a 3′-UTR as described in WO2017/036580, the disclosure ofWO2017/036580 relating to 3′-UTR sequences herewith incorporated byreference. Suitable 3′-UTRs are SEQ ID NOs: 152-204 of WO2017/036580, orfragments or variants of these sequences. In other embodiments, thenucleic acid comprises a 3′-UTR as described in WO2016/022914, thedisclosure of WO2016/022914 relating to 3′-UTR sequences herewithincorporated by reference. Particularly preferred 3′-UTRs are nucleicacid sequences according to SEQ ID NOs: 20-36 of WO2016/022914, orfragments or variants of these sequences.

In preferred embodiments, the RNA comprises at least one heterologous5′-UTR.

The terms “5′-untranslated region” or “5′-UTR” or “5′-UTR element” willbe recognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a part of an RNA molecule located 5′(i.e. “upstream”) of a coding sequence and which is not translated intoprotein. A 5′-UTR may be part of a nucleic acid located 5′ of the codingsequence. Typically, a 5′-UTR starts with the transcriptional start siteand ends before the start codon of the coding sequence. A 5′-UTR maycomprise elements for controlling gene expression, also calledregulatory elements. Such regulatory elements may be, e.g., ribosomalbinding sites, miRNA binding sites etc. The 5′-UTR may bepost-transcriptionally modified, e.g. by enzymatic orpost-transcriptional addition of a 5′-cap structure (e.g. for mRNA).

Preferably, the RNA comprises a 5′-UTR which may be derivable from agene that relates to an RNA with enhanced half-life (i.e. that providesa stable RNA).

In some embodiments, a 5′-UTR comprises one or more of a binding sitefor proteins that affect an RNA stability or RNA location in a cell, orone or more miRNA or binding sites for miRNAs (as defined above).

Accordingly, miRNA or binding sites for miRNAs as defined above may beremoved from the 5′-UTR or introduced into the 5′-UTR in order to tailorthe expression of the nucleic acid to desired cell types or tissues(e.g. muscle cells).

In preferred embodiments, the RNA comprises at least one heterologous5′-UTR, wherein the at least one heterologous 5′-UTR comprises a nucleicacid sequence derived from a 5′-UTR of gene selected from HSD1764,RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, andUBQLN2, or from a homolog, a fragment or variant of any one of thesegenes according to nucleic acid sequences being identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NOs: 231-252, 22870-22875 or afragment or a variant of any of these. Particularly preferred nucleicacid sequences in that context can be selected from published PCTapplication WO2019/077001A1, in particular, claim 9 of WO2019/077001A1.The corresponding 5′-UTR sequences of claim 9 of WO2019/077001A1 areherewith incorporated by reference (e.g., SEQ ID NOs: 1-20 ofWO2019/077001A1, or fragments or variants thereof).

In preferred embodiments, the RNA comprises a 5′-UTR derived from aRPL31 gene, wherein said 5′-UTR derived from a RPL31 gene comprises orconsists of a nucleic acid sequence being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 243, 244, 22872, 22873 or afragment or a variant thereof.

In other embodiments, the RNA comprises a 5′-UTR derived from a SLC7A3gene, wherein said 5′-UTR derived from a SLC7A3 gene comprises orconsists of a nucleic acid sequence being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 245, 246, 22874, 22875 or afragment or a variant thereof.

In particularly preferred embodiments, the RNA comprises a 5′-UTRderived from a HSD17B4 gene, wherein said 5′-UTR derived from a HSD17B4gene comprises or consists of a nucleic acid sequence being identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 231, 232, 22870,22871 or a fragment or a variant thereof.

In other embodiments, the RNA comprises a 5′-UTR which comprises orconsists of a nucleic acid sequence being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 22848-22867, 28522-28525 or afragment or a variant thereof.

In other embodiments, the RNA comprises a 5′-UTR as described inWO2013/143700, the disclosure of WO2013/143700 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WO2013/143700, orfragments or variants of these sequences. In other embodiments, thenucleic acid comprises a 5′-UTR as described in WO2016/107877, thedisclosure of WO2016/107877 relating to 5′-UTR sequences herewithincorporated by reference. Particularly preferred 5′-UTRs are nucleicacid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 ofWO2016/107877, or fragments or variants of these sequences. In otherembodiments, the nucleic acid comprises a 5′-UTR as described inWO2017/036580, the disclosure of WO2017/036580 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 ofWO2017/036580, or fragments or variants of these sequences. In otherembodiments, the nucleic acid comprises a 5′-UTR as described inWO2016/022914, the disclosure of WO2016/022914 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 ofWO2016/022914, or fragments or variants of these sequences.

Suitably, in preferred embodiments, the RNA comprises at least onecoding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2 operablylinked to a 3′-UTR and/or a 5′-UTR selected from the following5′UTR/3′UTR combinations (“also referred to UTR designs”): a-1(HSD1764/PSMB3), a-2 (NDUFA4/PSMB3), a-3 (SLC7A3/PSMB3), a-4(NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9),b-3 (HSD1764/RPS9), b-4 (HSD1764/CASP1), b-5 (NOSIP/COX6B1), c-1(NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4(NDUFA4/NDUFA1), c-5 (ATP5A1/PSMB3), d-1 (Rpl31/PSMB3), d-2(ATP5A1/CASP1), d-3 (SLC7A3/GNAS), d-4 (HSD1764/NDUFA1), d-5(Slc7a3/Ndufa1), e-1 (TUBB4B/RPS9), e-2 (RPL31/RPS9), e-3 (MP68/RPS9),e-4 (NOSIP/RPS9), e-5 (ATP5A1/RPS9), e-6 (ATP5A1/COX6B1), f-1(ATP5A1/GNAS), f-2 (ATP5A1/NDUFA1), f-3 (HSD1764/COX6B1), f-4(HSD1764/GNAS), f-5 (MP68/COX6B1), g-1 (MP68/NDUFA1), g-2(NDUFA4/CASP1), g-3 (NDUFA4/GNAS), g-4 (NOSIP/CASP1), g-5 (RPL31/CASP1),h-1 (RPL31/COX6B1), h-2 (RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4(S1c7a3/CASP1), h-5 (SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2(RPL32/ALB7), i-2 (RPL32/ALB7).

In particularly preferred embodiments, the RNA comprises at least onecoding sequence as specified herein encoding at least one antigenicprotein derived from SARS-CoV-2, wherein said coding sequence isoperably linked to a HSD17B4 5′-UTR and a PSMB3 3′-UTR (HSD17B4/PSMB3(UTR design a-1)).

It has been shown by the inventors that this embodiment is particularlybeneficial for induction an immune response against SARS-CoV-2. In thiscontext, it was shown that already one vaccination was sufficient toresult in virus-neutralizing antibody titers.

In further preferred embodiments, the nucleic acid comprises at leastone coding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto a SLC7A3 5′-UTR and a PSMB3 3′-UTR (SLC7A3/PSMB3 (UTR design a-3)).

In further preferred embodiments, the nucleic acid comprises at leastone coding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto a RPL31 5′-UTR and a RPS9 3′-UTR (RPL31/RPS9 (UTR design e-2)).

In some embodiments, the RNA may be monocistronic, bicistronic, ormulticistronic.

The term “monocistronic” will be recognized and understood by the personof ordinary skill in the art, and is e.g. intended to refer to a nucleicacid that comprises only one coding sequence. The terms “bicistronic”,or “multicistronic” as used herein will be recognized and understood bythe person of ordinary skill in the art, and are e.g. intended to referto a nucleic acid that may comprise two (bicistronic) or more(multicistronic) coding sequences.

In preferred embodiments, the RNA of the first aspect is monocistronic.

In other embodiments, the RNA is monocistronic and the coding sequenceof said nucleic acid encodes at least two different antigenic peptidesor proteins derived from a SARS-CoV-2. Accordingly, said coding sequencemay encode at least two, three, four, five, six, seven, eight and moreantigenic peptides or proteins derived from a SARS-CoV-2, linked with orwithout an amino acid linker sequence, wherein said linker sequence cancomprise rigid linkers, flexible linkers, cleavable linkers, or acombination thereof. Such constructs are herein referred to as“multi-antigen-constructs”.

In further embodiments, the RNA may be bicistronic or multicistronic andcomprises at least two coding sequences, wherein the at least two codingsequences encode two or more different antigenic peptides or proteinsderived from a SARS-CoV-2. Accordingly, the coding sequences in abicistronic or multicistronic nucleic acid suitably encodes distinctantigenic proteins or peptides as defined herein or immunogenicfragments or immunogenic variants thereof. Preferably, the codingsequences in said bicistronic or multicistronic constructs may beseparated by at least one IRES (internal ribosomal entry site) sequence.Thus, the term “encoding two or more antigenic peptides or proteins” maymean, without being limited thereto, that the bicistronic ormulticistronic nucleic acid encodes e.g. at least two, three, four,five, six or more (preferably different) antigenic peptides or proteinsof different SARS-CoV-2 isolates. Alternatively, the bicistronic ormulticistronic nucleic acid may encode e.g. at least two, three, four,five, six or more (preferably different) antigenic peptides or proteinsderived from the same SARS-CoV-2. In that context, suitable IRESsequences may be selected from the list of nucleic acid sequencesaccording to SEQ ID NOs: 1566-1662 of the patent applicationWO2017/081082, or fragments or variants of these sequences. In thiscontext, the disclosure of WO2017/081082 relating to IRES sequences isherewith incorporated by reference.

It has to be understood that, in the context of the invention, certaincombinations of coding sequences may be generated by any combination ofmonocistronic, bicistronic and multicistronic RNA constructs and/ormulti-antigen-constructs to obtain a nucleic acid set encoding multipleantigenic peptides or proteins as defined herein.

In preferred embodiments, the A/U (A/T) content in the environment ofthe ribosome binding site of the RNA may be increased compared to theA/U (A/T) content in the environment of the ribosome binding site of itsrespective wild type or reference RNA. This modification (an increasedA/U (A/T) content around the ribosome binding site) increases theefficiency of ribosome binding to the RNA. An effective binding of theribosomes to the ribosome binding site in turn has the effect of anefficient translation the RNA.

Accordingly, in a particularly preferred embodiment, the RNA comprises aribosome binding site, also referred to as “Kozak sequence”, identicalto or at least 80%, 85%, 90%, 95% identical to any one of the sequencesSEQ ID NOs: 180, 181, 22845-22847, or fragments or variants thereof.

In preferred embodiments, the RNA comprises at least one poly(N)sequence, e.g. at least one poly(A) sequence, at least one poly(U)sequence, at least one poly(C) sequence, or combinations thereof.

In preferred embodiments, the RNA of the invention comprises at leastone poly(A) sequence.

The terms “poly(A) sequence”, “poly(A) tail” or “3′-poly(A) tail” asused herein will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to be a sequence of adenosinenucleotides, typically located at the 3′-end of a linear RNA (or in acircular RNA), of up to about 1000 adenosine nucleotides. Preferably,said poly(A) sequence is essentially homopolymeric, e.g. a poly(A)sequence of e.g. 100 adenosine nucleotides has essentially the length of100 nucleotides. In other embodiments, the poly(A) sequence isinterrupted by at least one nucleotide different from an adenosinenucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotidesmay have a length of more than 100 nucleotides (comprising 100 adenosinenucleotides and in addition said at least one nucleotide—or a stretch ofnucleotides—different from an adenosine nucleotide).

The poly(A) sequence may comprise about 10 to about 500 adenosinenucleotides, about 10 to about 200 adenosine nucleotides, about 40 toabout 200 adenosine nucleotides, or about 40 to about 150 adenosinenucleotides. Suitably, the length of the poly(A) sequence may be atleast about or even more than about 10, 50, 64, 75, 100, 200, 300, 400,or 500 adenosine nucleotides. In certain embodiments the RNA comprisesat least one poly(A) sequence comprising 30 to 200 adenosinenucleotides, wherein the 3′ terminal nucleotide of said RNA is anadenosine.

In preferred embodiments, the RNA of the invention comprises at leastone poly(A) sequence comprising about 30 to about 200 adenosinenucleotides. In particularly preferred embodiments, the poly(A) sequencecomprises about 64 adenosine nucleotides (A64). In particularlypreferred embodiments, the poly(A) sequence comprises about 100adenosine nucleotides (A100). In other embodiments, the poly(A) sequencecomprises about 150 adenosine nucleotides.

In further embodiments, the RNA of the invention comprises at least onepoly(A) sequence comprising about 100 adenosine nucleotides, wherein thepoly(A) sequence is interrupted by non-adenosine nucleotides, preferablyby 10 non-adenosine nucleotides (A30-N10-A70).

The poly(A) sequence as defined herein may be located directly at the 3′terminus of the RNA, preferably directly at the 3′ terminus of an RNA.

In preferred embodiments, the 3′-terminal nucleotide (that is the last3′-terminal nucleotide in the polynucleotide chain) is the 3′-terminal Anucleotide of the at least one poly(A) sequence. The term “directlylocated at the 3′ terminus” has to be understood as being locatedexactly at the 3′ terminus—in other words, the 3′ terminus of thenucleic acid consists of a poly(A) sequence terminating with an Anucleotide.

It has been shown by the inventors that this embodiment is particularlybeneficial for induction an immune response against SARS-CoV-2. In thiscontext, it was shown that already one vaccination was sufficient toresult in virus-neutralizing antibody titers.

In a particularly preferred embodiment, the RNA sequence comprises apoly(A) sequence of at least 70 adenosine nucleotides, wherein the3′-terminal nucleotide is an adenosine nucleotide.

In this context it has been shown that ending on an adenosine nucleotidedecreases the induction of IFNalpha by the RNA vaccine. This isparticularly important as the induction of IFNalpha is thought to be themain factor for induction of fever in vaccinated subjects, which ofcourse has to be avoided.

In preferred embodiments, the poly(A) sequence of the RNA is obtainedfrom a DNA template during RNA in vitro transcription. In otherembodiments, the poly(A) sequence is obtained in vitro by common methodsof chemical synthesis without being necessarily transcribed from a DNAtemplate. In other embodiments, poly(A) sequences are generated byenzymatic polyadenylation of the RNA (after RNA in vitro transcription)using commercially available polyadenylation kits and correspondingprotocols known in the art, or alternatively, by using immobilizedpoly(A)polymerases e.g. using a methods and means as described inWO2016/174271, the entire contents of which are hereby incorporated byreference.

In some embodiments, the RNA comprises a poly(A) sequence obtained byenzymatic polyadenylation, wherein the majority of RNA moleculescomprise about 100 (+/−20) to about 500 (+/−50), preferably about 250(+/−20) adenosine nucleotides.

In other embodiments, the RNA comprises a poly(A) sequence derived froma template DNA and at least one additional poly(A) sequence generated byenzymatic polyadenylation, e.g. as described in WO2016/091391, theentire contents of which are hereby incorporated by reference.

In further embodiments, the RNA comprises at least one poly(C) sequence.

The term “poly(C) sequence” as used herein is intended to be a sequenceof cytosine nucleotides of up to about 200 cytosine nucleotides. Inpreferred embodiments, the poly(C) sequence comprises about 10 to about200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides,about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosinenucleotides, or about 10 to about 40 cytosine nucleotides. In aparticularly preferred embodiment, the poly(C) sequence comprises about30 cytosine nucleotides.

In preferred embodiments, the RNA of the invention comprises at leastone histone stem-loop (hSL).

The term “histone stem-loop” (abbreviated as “hSL” in e.g. the sequencelisting) is intended to refer to a nucleic acid sequences that form astem-loop secondary structure predominantly found in histone mRNAs.

Histone stem-loop sequences/structures may suitably be selected fromhistone stem-loop sequences as disclosed in WO2012/019780, the entirecontents of which are hereby incorporated by reference, the disclosurerelating to histone stem-loop sequences/histone stem-loop structuresincorporated herewith by reference. A histone stem-loop sequence thatmay be used within the present invention may preferably be derived fromformulae (I) or (II) of WO2012/019780. According to a further preferredembodiment, the RNA comprises at least one histone stem-loop sequencederived from at least one of the specific formulae (Ia) or (IIa) of thepatent application WO2012/019780.

In preferred embodiments, the RNA of the invention comprises at leastone histone stem-loop, wherein said histone stem-loop (hSL) comprises orconsists a nucleic acid sequence identical or at least 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs:178 or 179, or fragments or variants thereof.

In other embodiments, the RNA does not comprise a histone stem-loop asdefined herein.

In various embodiments, the RNA comprises a 3′-terminal sequenceelement. Said 3′-terminal sequence element comprises a poly(A) sequenceand optionally a histone-stem-loop sequence. Accordingly, the RNA of theinvention comprises at least one 3′-terminal sequence element comprisingor consisting of a nucleic acid sequence being identical or at least70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NOs: 254, 22893, 22903, 26997, 26999, 28529, 28531, 28533,28535, 28537, 28539 or a fragment or variant thereof.

In preferred embodiments, the RNA comprises a 3′-terminal sequenceelement. Said 3′-terminal sequence element comprises a poly(A) sequence.Accordingly, the nucleic acid of the invention comprises at least one3′-terminal sequence element comprising or consisting of a nucleic acidsequence being identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 254, 22903, 26999,28531, 28525, 28539 or a fragment or variant thereof.

In preferred embodiments, the RNA comprises a 3′-terminal sequenceelement. Said 3′-terminal sequence element comprises a poly(A) sequenceand a histone-stem-loop sequence. Accordingly, the nucleic acid of theinvention comprises at least one 3′-terminal sequence element comprisingor consisting of a nucleic acid sequence being identical or at least70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NOs: 254, 22893, 26997, 28529, 28533, 28537 or a fragment orvariant thereof.

In various embodiments, the RNA may comprise a 5′-terminal sequenceelement according to SEQ ID NOs: 176, 177 or 22840-22844, or a fragmentor variant thereof.

In further embodiments, the RNA may comprise a 5′-terminal sequenceelement according to SEQ ID NOs: 176, 177 or 22840-22844 or a fragmentor variant thereof. Such a 5′-terminal sequence element comprises e.g. abinding site for T7 RNA polymerase. Further, the first nucleotide ofsaid 5′-terminal start sequence may preferably comprise a 2′Omethylation, e.g. 2′O methylated guanosine or a 2′O methylatedadenosine.

In preferred embodiments, the comprises at least one heterologous 5′-UTRthat comprises or consists of a nucleic acid sequence derived from a5′-UTR from HSD17B4 and at least one heterologous 3′-UTR comprises orconsists of a nucleic acid sequence derived from a 3′-UTR of PSMB3. Incertain embodiments, the 5′-UTR from HSD17B4 is at least about 95%, 96%,97%, 98% to 99% identical to to SEQ ID NO: 232. In some embodiments, the3′-UTR of PSMB3 is at least about 95%, 96%, 97%, 98% to 99% identical toto SEQ ID NO: 254. In especially preferred embodiments the RNAcomprises, from 5′ to 3′: i) 5′-cap1 structure; ii) 5′-UTR derived froma 5′-UTR of a HSD17B4 gene, preferably according to SEQ ID NO: 232; iii)the at least one coding sequence (encoding a SARS-CoV Spike antigen ofthe embodiments); iv) 3′-UTR derived from a 3′-UTR of a PSMB3 gene,preferably according to SEQ ID NO: 254; v) optionally, a histonestem-loop sequence; and vi) poly(A) sequence comprising about 100 Anucleotides, wherein the 3′ terminal nucleotide of said RNA is anadenosine.

Preferably, the RNA comprises about 50 to about 20000 nucleotides, orabout 500 to about 10000 nucleotides, or about 1000 to about 10000nucleotides, or preferably about 1000 to about 5000 nucleotides, or evenmore preferably about 2000 to about 5000 nucleotides.

According to particularly preferred embodiments, the RNA is a codingRNA. In preferred embodiments, the coding RNA may be selected from anmRNA, a (coding) self-replicating RNA, a (coding) circular RNA, a(coding) viral RNA, or a (coding) replicon RNA.

In other embodiments, the coding RNA is a circular RNA. As used herein,“circular RNA” or “circRNAs” have to be understood as a circularpolynucleotide constructs that encode at least one antigenic peptide orprotein as defined herein. Preferably, such a circRNA is a singlestranded RNA molecule. In preferred embodiments, said circRNA comprisesat least one coding sequence encoding at least one antigenic proteinfrom a SARS-CoV-2 coronavirus, or an immunogenic fragment or animmunogenic variant thereof.

In further embodiments, the coding RNA is a replicon RNA. The term“replicon RNA” will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to be an optimizedself-replicating RNA. Such constructs may include replicase elementsderived from e.g. alphaviruses (e.g. SFV, SIN, VEE, or RRV) and thesubstitution of the structural virus proteins with the nucleic acid ofinterest (that is, the coding sequence encoding an antigenic peptide orprotein of a SARS-CoV-2 coronavirus). Alternatively, the replicase maybe provided on an independent coding RNA construct or a coding DNAconstruct. Downstream of the replicase may be a sub-genomic promoterthat controls replication of the replicon RNA.

In particularly preferred embodiments, the at least one nucleic acid isnot a replicon RNA or a self-replicating RNA.

In particularly preferred embodiments, the RNA of the invention is anmRNA.

Preferably, the mRNA does not comprise a replicase element (e.g. anucleic acid encoding a replicase).

The terms “RNA” and “mRNA” will be recognized and understood by theperson of ordinary skill in the art, and are e.g. intended to be aribonucleic acid molecule, i.e. a polymer consisting of nucleotides.These nucleotides are usually adenosine-monophosphate,uridine-monophosphate, guanosine-monophosphate andcytidine-monophosphate monomers which are connected to each other alonga so-called backbone. The backbone is formed by phosphodiester bondsbetween the sugar, i.e. ribose, of a first and a phosphate moiety of asecond, adjacent monomer. The specific succession of the monomers iscalled the RNA-sequence. The mRNA (messenger RNA) provides thenucleotide coding sequence that may be translated into an amino-acidsequence of a particular peptide or protein.

In the context of the invention, the RNA, preferably the mRNA, providesat least one coding sequence encoding an antigenic protein from aSARS-CoV-2 spike protein as defined herein that is translated into a(functional) antigen after administration (e.g. after administration toa subject, e.g. a human subject).

In preferred embodiments, the RNA, preferably the mRNA is suitable for aSARS-CoV-2 vaccine, preferably a SARS-CoV-2 vaccine against at least oneof the following SARS-CoV-2 isolates: C.1.2 (South Africa), B.1.1.529(Omicron, South Africa) (including BA.1_v1, BA.1_v0, B.1.1.529, BA.2,BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand), B.1.619(Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1 (India),AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India), B.1.617.3(India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK), P.1 (Gamma,Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525 (Eta,Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US), A.23.1(Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta,Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621(Mu, Columbia).

In particularly preferred embodiments, the RNA, preferably the mRNA issuitable for a SARS-CoV-2 vaccine, preferably a SARS-CoV-2 vaccineagainst B.1.351 (South Africa) or B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5).

Suitably, the RNA may be modified by the addition of a 5′-cap structure,which preferably stabilizes the RNA and/or enhances expression of theencoded antigen and/or reduces the stimulation of the innate immunesystem (after administration to a subject). A 5′-cap structure is ofparticular importance in embodiments where the RNA is a linear codingRNA, e.g. a linear mRNA or a linear coding replicon RNA.

Accordingly, in preferred embodiments, the RNA, in particular the mRNAcomprises a 5′-cap structure, preferably cap0, cap1, cap2, a modifiedcap0, or a modified cap1 structure.

The term “5′-cap structure” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a 5′ modified nucleotide, particularly a guaninenucleotide, positioned at the 5′-end of an RNA, e.g. an mRNA.Preferably, the 5′-cap structure is connected via a 5′-5′-triphosphatelinkage to the RNA.

5′-cap structures which may be suitable in the context of the presentinvention are cap0 (methylation of the first nucleobase, e.g. m7GpppN),cap1 (additional methylation of the ribose of the adjacent nucleotide ofm7GpppN), cap2 (additional methylation of the ribose of the 2ndnucleotide downstream of the m7GpppN), cap3 (additional methylation ofthe ribose of the 3rd nucleotide downstream of the m7GpppN), cap4(additional methylation of the ribose of the 4th nucleotide downstreamof the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

A 5′-cap (cap0 or cap1) structure may be formed in chemical RNAsynthesis or in RNA in vitro transcription (co-transcriptional capping)using cap analogues.

The term “cap analogue” as used herein will be recognized and understoodby the person of ordinary skill in the art, and is e.g. intended torefer to a non-polymerizable di-nucleotide or tri-nucleotide that hascap functionality in that it facilitates translation or localization,and/or prevents degradation of a nucleic acid molecule, particularly ofan RNA molecule, when incorporated at the 5′-end of the nucleic acidmolecule. Non-polymerizable means that the cap analogue will beincorporated only at the 5′-terminus because it does not have a 5′triphosphate and therefore cannot be extended in the 3′-direction by atemplate-dependent polymerase, particularly, by template-dependent RNApolymerase. Examples of cap analogues include, but are not limited to, achemical structure selected from the group consisting of m7GpppG,m7GpppA, m7GpppC; unmethylated cap analogues (e.g. GpppG); dimethylatedcap analogue (e.g. m2,7GpppG), trimethylated cap analogue (e.g.m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g. m7Gpppm7G),or anti reverse cap analogues (e.g. ARCA; m7,2′OmeGpppG, m7,2′dGpppG,m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives).Further cap analogues have been described previously (WO2008/016473,WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475). Furthersuitable cap analogues in that context are described in WO2017/066793,WO2017/066781, WO2017/066791, WO2017/066789, WO2017/053297,WO2017/066782, WO2018/075827 and WO2017/066797 wherein the disclosuresreferring to cap analogues are incorporated herewith by reference.

In some embodiments, a modified cap1 structure is generated usingtri-nucleotide cap analogue as disclosed in WO2017/053297,WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789,WO2017/066782, WO2018/075827 and WO2017/066797, the entire contents ofthe aforementioned PCT applications are hereby incorporated byreference. In particular, any cap structures derivable from thestructure disclosed in claim 1-5 of WO2017/053297 may be suitably usedto co-transcriptionally generate a modified cap1 structure. Further, anycap structures derivable from the structure defined in claim 1 or claim21 of WO2018/075827 may be suitably used to co-transcriptionallygenerate a modified cap1 structure.

In preferred embodiments, the RNA, in particular the mRNA comprises acap1 structure.

In preferred embodiments, the 5′-cap structure may suitably be addedco-transcriptionally using tri-nucleotide cap analogue as defined hereinin an RNA in vitro transcription reaction as defined herein.

In preferred embodiments, the cap1 structure of the coding RNA of theinvention is formed using co-transcriptional capping usingtri-nucleotide cap analogues m7G(5′)ppp(5′)(2′OMeA)pG orm7G(5′)ppp(5′)(2′OMeG)pG. A preferred cap1 analogues in that context ism7G(5′)ppp(5′)(2′OMeA)pG.

In other preferred embodiments, the cap1 structure of the RNA of theinvention is formed using co-transcriptional capping usingtri-nucleotide cap analogue 3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG.

In other embodiments, a cap0 structure of the RNA of the invention isformed using co-transcriptional capping using cap analogue3′OMe-m7G(5′)ppp(5′)G.

In other embodiments, the 5′-cap structure is formed via enzymaticcapping using capping enzymes (e.g. vaccinia virus capping enzymesand/or cap-dependent 2′-O methyltransferases) to generate cap0 or cap1or cap2 structures. The 5′-cap structure (cap0 or cap1) may be addedusing immobilized capping enzymes and/or cap-dependent 2′-Omethyltransferases using methods and means disclosed in WO2016/193226,the entire content of which is hereby incorporated by reference.

In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA(species) comprises a cap1 structure as determined using a cappingassay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%,3%, 2%, 1% of the RNA (species) does not comprise a cap1 structure asdetermined using a capping assay. In other preferred embodiments, about70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises a cap0structure as determined using a capping assay. In preferred embodiments,less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (species)does not comprise a cap0 structure as determined using a capping assay.

The term “RNA species” is not restricted to mean “one single molecule”but is understood to comprise an ensemble of essentially identical RNAmolecules. Accordingly, it may relate to a plurality of essentiallyidentical (coding) RNA molecules.

For determining the presence/absence of a cap0 or a cap1 structure, acapping assay as described in published PCT application WO2015/101416,the entire content of which is hereby incorporated by reference; inparticular, as described in claims 27 to 46 of published PCT applicationWO2015/101416 can be used. Other capping assays that may be used todetermine the presence/absence of a cap0 or a cap1 structure of an RNAare described in PCT/EP2018/08667, or published PCT applicationsWO2014/152673 and WO2014/152659, the entire content of theaforementioned PCT applications are hereby incorporated by reference.

In preferred embodiments, the RNA comprises an m7G(5′)ppp(5′)(2′OMeA)cap structure. In such embodiments, the coding RNA comprises a5′-terminal m7G cap, and an additional methylation of the ribose of theadjacent nucleotide of m7GpppN, in that case, a 2′O methylatedAdenosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA(species) comprises such a cap1 structure as determined using a cappingassay.

In other preferred embodiments, the RNA comprises anm7G(5′)ppp(5′)(2′OMeG) cap structure. In such embodiments, the codingRNA comprises a 5′-terminal m7G cap, and an additional methylation ofthe ribose of the adjacent nucleotide, in that case, a 2′O methylatedguanosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the codingRNA (species) comprises such a cap1 structure as determined using acapping assay.

Accordingly, the first nucleotide of said RNA or mRNA sequence, that is,the nucleotide downstream of the m7G(5′)ppp structure, may be a 2′Omethylated guanosine or a 2′O methylated adenosine.

According to some embodiments, the RNA is a modified RNA, wherein themodification refers to chemical modifications comprising backbonemodifications as well as sugar modifications or base modifications.

A modified RNA may comprise nucleotide analogues/modifications, e.g.backbone modifications, sugar modifications or base modifications. Abackbone modification in the context of the invention is a modification,in which phosphates of the backbone of the nucleotides of the RNA arechemically modified. A sugar modification in the context of theinvention is a chemical modification of the sugar of the nucleotides ofthe RNA. Furthermore, a base modification in the context of theinvention is a chemical modification of the base moiety of thenucleotides of the RNA. In this context, nucleotide analogues ormodifications are preferably selected from nucleotide analogues whichare applicable for transcription and/or translation.

In particularly preferred embodiments, the nucleotideanalogues/modifications which may be incorporated into a modified RNA asdescribed herein are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl-inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate,pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,1-carboxymethyl-pseudouridine, 5-propynyl-uridine,1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine,5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine,5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine,5′-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine,alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, alpha -thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

In some embodiments, the at least one modified nucleotide is selectedfrom pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.

In some embodiments, 100% of the uracil in the coding sequence asdefined herein have a chemical modification, preferably a chemicalmodification is in the 5-position of the uracil.

Particularly preferred in the context of the invention are pseudouridine(ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and5-methoxyuridine.

In some embodiments, however, the RNA of the invention does not includeany N1-methylpseudouridine (m1ψ) substituted positions. In furtheraspects, the RNA of the embodiments does not include any pseudouridine(ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and5-methoxyuridine substituted position. In still further embodiments, theRNA of the invention comprises a coding sequence that consists only ofG, C, A and U nucleotides.

Incorporating modified nucleotides such as pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 5-methylcytosine, and/or 5-methoxyuridineinto the coding sequence of the RNA may be advantageous as unwantedinnate immune responses (upon administration of the coding RNA or thevaccine) may be adjusted or reduced (if required).

In some embodiments, the RNA comprises at least one coding sequenceencoding a SARS-CoV-2 antigenic protein as defined herein, wherein saidcoding sequence comprises at least one modified nucleotide selected frompseudouridine (ψ) and N1-methylpseudouridine (m1ψ), preferably whereinall uracil nucleotides are replaced by pseudouridine (ψ) nucleotidesand/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA does not compriseN1-methylpseudouridine (m1ψ) substituted positions. In furtherembodiments, the RNA does not comprise pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridinesubstituted position.

In preferred embodiments, the RNA comprises a coding sequence thatconsists only of G, C, A and U nucleotides and therefore does notcomprise modified nucleotides (except of the 5′ terminal cap structure,e.g. cap1)

Nucleic Acid, Preferably mRNA Constructs Suitable for a CoronavirusVaccine:

In various embodiments the RNA, preferably the mRNA comprises,preferably in 5′- to 3′-direction, the following elements:

-   -   A) 5′-cap structure, preferably as specified herein;    -   B) 5′-terminal start element, preferably as specified herein;    -   C) optionally, a 5′-UTR, preferably as specified herein;    -   D) a ribosome binding site, preferably as specified herein;    -   E) at least one coding sequence, preferably as specified herein;    -   F) 3′-UTR, preferably as specified herein;    -   G) optionally, poly(A) sequence, preferably as specified herein;    -   H) optionally, poly(C) sequence, preferably as specified herein;    -   I) optionally, histone stem-loop preferably as specified herein;    -   J) optionally, 3′-terminal sequence element, preferably as        specified herein.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 116, 136, 137, 146,        22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781,        22783, 22785, 23089-23148, 23150-23184, 27110-27247,        28589-28637, 28916, 28921-28924 or fragments or variants        thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 116, 136, 137, 146,        22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779, 22781,        22783, 22785, 23089-23148, 23150-23184, 27110-27247,        28589-28637, 28916, 28921-28924 or fragments or variants        thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 27118, 27141,        27164, 27187, 27210, 27233, 28601-28606 or fragments or variants        thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 27118, 27141,        27164, 27187, 27210, 27233, 28601-28606 or fragments or variants        thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 27118, 27141,        27164, 27187, 27210, 27233 or fragments or variants thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 27118, 27141,        27164, 27187, 27210, 27233 or fragments or variants thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 28590-28593,        28921-28924 or fragments or variants thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   -   A) cap1 structure as defined herein;    -   B) 5′-UTR derived from a HSD17B4 gene as defined herein,        preferably according to SEQ ID NOs: 231 or 232;    -   C) coding sequence selected from SEQ ID NOs: 28590-28593,        28921-28924 or fragments or variants thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined        herein, preferably according to SEQ ID NOs: 253 or 254;    -   F) poly(A) sequence comprising about 100 A nucleotides,        preferably representing the 3′ terminus.

Preferred RNA sequences, preferably mRNA sequences of the invention areprovided in Table 2. Therein, each row represents a specific suitableSARS-CoV-2 construct of the invention, wherein the description of theSARS-CoV-2 backbone construct is indicated in column A (Col A) of Table2 and the corresponding RNA sequences, in particular mRNA sequencescomprising preferred coding sequences are provided in columns C-G (Col.C-G). Table 2a, provides RNA sequences with HSD1764/PSMB3-hSL-A100(a-1). Table 2b with HSD1764/PSMB3-A100 (a-1).

TABLE 2a Nucleic acid, preferably mRNA constructs suitable for a vaccineCol A Col B Col C Col D Col E Col F Col G S_stab_PP(K986P_V987P_D614G)22792, 23536 23554 23572 23590 23608 28737S_stab_PP(K986P_V987P_A222V_D614G) 22794, 23537 23555 23573 23591 2360928738 S_stab_PP(K986P_V987P_N439K_D614G) 22796, 23538 23556 23574 2359223610 28739 S_stab_PP(K986P_V987P_S477N_D614G) 22798, 23539 23557 2357523593 23611 28740 S_stab_PP(K986P_V987P_N501Y_D614G) 22800, 23540 2355823576 23594 23612 28741 S_stab_PP(K986P_V987P_H69del_V70del_D614G)22802, 23541 23559 23577 23595 23613 28742S_stab_PP(K986P_V987P_Y453F_D614G) 22804, 23542 23560 23578 23596 2361428743 S_stab_PP(K986P_V987P_D614G_LI692V) 22806, 23543 23561 23579 2359723615 28744 S_stab_PP(K986P_V987P_D614G_M1229I) 22808, 23544 23562 2358023598 23616 28745 S_stab_PP(K986P_V987P_H69del_V70del_A222V_(—) 22810,23545 23563 23581 23599 23617 Y453F_S477N_D614G_I692V) 28746S_stab_PP(K986P_V987P_H69del_V70del_Y453F_(—) 22812, 23546 23564 2358223600 23618 D614G_I692V_M1229I) 28747S_stab_PP(K986P_V987P_H69del_V70del_Y144del_(—) 23529, 23547 23565 2358323601 23619 N501Y_A570D_D614G_P681H_T716I_S982A_(—) 28748 D1118H)S_stab_PP(K986P_V987P_K417N_E484K_N501Y_(—) 23530, 23548 23566 2358423602 23620 D614G) 28749 S_stab_PP(K986P_V987P_L18F_D80A_D215G_(—)23531, 23549 23567 23585 23603 23621L242del_A243del_L244del_R246I_K417N_E484K_(—) 28750 N501Y_D614G_A701V)S_stab_PP(K986P_V987P_E484K_D614G) 23532, 23550 23568 23586 23604 2362228751 S_stab_PP(K986P_V987P_L18F_T20N_P26S_(—) 23533, 23551 23569 2358723605 23623 D138Y_R190S_K417T_E484K_N501Y_D614G_(—) 28752 H655Y_T1027I)S_stab_PP(K986P_V987P_S13I_W152C_L452R_D614G) 23534, 23552 23570 2358823606 23624 28753 S_stab_PP(K986P_V987P_H69del_V70del_Y144del_(—) 27386,27409 27432 27455 27478 27501 E484K_N501Y_A570D_D614G_P681H_T716I_(—)28754 S982A_D1118H) S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_(—)27387, 27410 27433 27456 27479 27502A243del_L244del_K417N_E484K_N501Y_D614G_A701V) 28755S_stab_PP(K986P_V987P_Q52R_A67V_H69del_(—) 27388, 27411 27434 2745727480 27503 V70del_Y144del_E484K_D614G_Q677H_F888L) 28756S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 27389, 27412 27435 2745827481 27504 Y144del_E484K_D614G_Q677H_F888L) 28757S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_(—) 27390, 27413 27436 2745927482 27505 R190S_K417T_E484K_N501Y_D614G_H655Y_(—) 28758 T1027I_V1176F)S_stab_PP(K986P_V987P_E484K_D614G_V1176F) 27391, 27414 27437 27460 2748327506 28759 S_stab_PP(K986P_V987P_L452R_D614G_P681R) 27392, 27415 2743827461 27484 27507 28760 S_stab_PP(K986P_V987P_E154K_L452R_E484Q_(—)27393, 27416 27439 27462 27485 27508 D614G_P681R_Q1071H) 28761S_stab_PP(K986P_V987P_T19R_F157del_R158del_(—) 27394, 27417 27440 2746327486 27509 L452R_T478K_D614G_P681R_D950N) 28762S_stab_PP(K986P_V987P_G75V_T76I_R246del_(—) 27395, 27418 27441 2746427487 27510 S247del_Y248del_L249del_T250del_P251del_(—) 28763G252del_L452Q_F490S_D614G_T859N)S_stab_PP(K986P_V987P_H69del_V70del_N439K_(—) 27396, 27419 27442 2746527488 27511 D614G) 28764 S_stab_PP(K986P_V987P_L5F_T95I_D253G_E484K_(—)27397, 27420 27443 27466 27489 27512 D614G_A701V) 28765S_stab_PP(K986P_V987P_L5F_T95I_D253G_S477N_(—) 27398, 27421 27444 2746727490 27513 D614G_Q957R) 28766S_stab_PP(K986P_V987P_F157L_V367F_Q613H_(—) 27399, 27422 27445 2746827491 27514 P681R) 28767 S_stab_PP(K986P_V987P_S254F_D614G_P681R_(—)27400, 27423 27446 27469 27492 27515 G769V) 28768S_stab_PP(K986P_V987P_P26S_H69del_V70del_(—) 27401, 27424 27447 2747027493 27516 V126A_Y144del_L242del_A243del_L244del_H245Y_(—) 28769S477N_E484K_D614G_P681H_T1027I_D1118H)S_stab_PP(K986P_V987P_T95I_Y144T_Y145S_(—) 27402, 27425 27448 2747127494 27517 ins145N_R346K_E484K_N501Y_D614G_P681H_(—) 28770 D950N)S_stab_PP(K986P_V987P_ins214TDR_Q414K_(—) 27403, 27426 27449 27472 2749527518 N450K_D614G_T716I) 28771S_stab_PP(K986P_V987P_T478K_D614G_P681H_(—) 27404, 27427 27450 2747327496 27519 T732A) 28772 S_stab_PP(K986P_V987P_E484K_N501Y_D614G_(—)27405, 27428 27451 27474 27497 27520 P681H_E1092K_H1101Y_V1176F) 28773S_stab_PP(K986P_V987P_H66D_G142V_Y144del_(—) 27406, 27429 27452 2747527498 27521 Y145del_D215G_V483A_D614G_H655Y_G669S_(—) 28774Q949R_N1187D) S_stab_PP(K986P_V987P_Y144del_L452R_T478K_(—) 27407, 2743027453 27476 27499 27522 P681R) 28775S_stab_PP(K986P_V987P_T19R_Y144del_Y145del_(—) 27408, 27431 27454 2747727500 27523 L452R_T478K_D614G_P681R) 28776S_stab_PP(K986P_V987P_P9L_C136F_Y144del_(—) 28638,R190S_D215G_L242del_A243del_Y449H_E484K_(—) 28777N501Y_D614G_H655Y_N679K_T716I_T859N)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28639,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28778L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_(—)P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v1)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28640,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28779L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)K417N_N440K_G446S_S477N_T478K_E484A_(—)Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_(—)H655Y_N679K_P681H_N764K_D796Y_N856K_(—) Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v0) S_stab_PP(K986P_V987P_(—) 28641,A67V_T95I_G339D_S371L_S373P_S375F_S477N_(—) 28780T478K_E484A_Q493R_G496S_Q498R_N501Y_(—)Y505H_T547K_D614G_H655Y_N679K_P681H_(—) D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_B.1.1.529) S_stab_PP(K986P_V987P_(—) 28642,T19I_L24del_P25del_P26del_A27S_G142D_V213G_(—) 28781G339D_S371F_S373P_S375F_T376A_D405N_S477N_(—)T478K_E484A_Q493R_Q498R_N501Y_Y505H_D614G_(—)H655Y_N679K_P681H_D796Y_Q954H_N969K); S_stab_PP(K986P_V987P_BA.2)S_stab_PP(K986P_V987P_G75V_T76I_R246del_(—) 28643,S247del_Y248del_L249del_T250del_P251del_(—) 28782G252del_D253N_L452Q_F490S_D614G_T859N)S_stab_PP(K986P_V987P_T95I_Y144S_Y145N_(—) 28644,R346K_E484K_N501Y_D614G_P681H_D950N) 28783S_stab_PP(K986P_V987P_S12F_H69del_V70del_(—) 28645,W152R_R346S_L452R_D614G_Q677H_A899S) 28784S_stab_PP(K986P_V987P_I210T_N440K_E484K_(—) 28646,D614G_D936N_S939F_T1027I) 28785S_stab_PP(K986P_V987P_W152L_E484K_D614G_(—) 28647, G769V) 28786S_stab_PP(K986P_V987P_T20I_R357K_D614G) 28648, 28787S_stab_PP(K986P_V987P_T95I_Y144del_E484K_(—) 28649, D614G_P681H_D796H)28788 S_stab_PP(K986P_V987P_T19R_G142D_E156G_(—) 28650,F157del_R158del_L452R_T478K_D614G_P681R_(—) 28789 D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28651,E156G_F157del_R158del_W258L_K417N_L452R_(—) 28790T478K_D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_V70F_G142D_(—)28652, E156G_F157del_R158del_A222V_K417N_L452R_(—) 28791T478K_D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—)28653, E156G_F157del_R158del_L452R_T478K_D614G_(—) 28792 P681R_D950N)S_stab_PP(K986P_V987P_T19R_E156G_F157del_(—) 28654,R158del_L452R_T478K_D614G_P681R_D950N) 28793S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28655,Y145H_E156G_F157del_R158del_A222V_L452R_(—) 28794T478K_D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_L452R_E484Q_(—)28656, D614G_P681R_D950N) 28795S_stab_PP(K986P_V987P_L452R_D614G_P681R_(—) 28657,R682del_R683del_A684del_R685del) 28796S_stab_PP(K986P_V987P_E154K_L452R_E484Q_(—) 28658,D614G_P681R_R682del_R683del_A684del_(—) 28797 R685del_Q1071H)S_stab_PP(K986P_V987P_T19R_F157del_R158del_(—) 28659,L452R_T478K_D614G_P681R_R682del_R683del_(—) 28798 A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_G142D_E156G_(—) 28660,F157del_R158del_L452R_T478K_D614G_P681R_(—) 28799R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28661,E156G_F157del_R158del_W258L_K417N_L452R_(—) 28800T478K_D614G_P681R_R682del_R683del_A684del_(—) R685del_D950N)S_stab_PP(K986P_V987P_T19R_V70F_G142D_(—) 28662,E156G_F157del_R158del_A222V_K417N_L452R_(—) 28801T478K_D614G_P681R_R682del_R683del_A684del_(—) R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28663,E156G_F157del_R158del_L452R_T478K_D614G_(—) 28802P681R_R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_E156G_F157del_(—) 28664,R158del_L452R_T478K_D614G_P681R_R682del_(—) 28803R683del_A684del_R685del_D950N) S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—)28665, Y145H_E156G_F157del_R158del_A222V_L452R_(—) 28804T478K_D614G_P681R_R682del_R683del_A684del_(—) R685del_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484Q_(—) 28666,D614G_P681R_R682del_R683del_A684del_(—) 28805 R685del_D950N)S_stab_PP(K986P_V987P_L452R_E484K_D614G_(—) 28667, P681R) 28806S_stab_PP(K986P_V987P_E154K_L452R_E484K_(—) 28668, D614G_P681R_Q1071H)28807 S_stab_PP(K986P_V987P_T19R_F157del_R158del_(—) 28669,L452R_T478K_E484K_D614G_P681R_D950N) 28808S_stab_PP(K986P_V987P_T19R_G142D_E156G_(—) 28670,F157del_R158del_L452R_T478K_E484K_D614G_(—) 28809 P681R_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28671,E156G_F157del_R158del_W258L_K417N_L452R_(—) 28810T478K_E484K_D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_V70F_G142D_(—)28672, E156G_F157del_R158del_A222V_K417N_L452R_(—) 28811T478K_E484K_D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—)28673, E156G_F157del_R158del_L452R_T478K_E484K_(—) 28812D614G_P681R_D950N) S_stab_PP(K986P_V987P_T19R_E156G_F157del_(—) 28674,R158del_L452R_T478K_E484K_D614G_P681R_(—) 28813 D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28675,Y145H_E156G_F157del_R158del_A222V_L452R_(—) 28814T478K_E484K_D614G_P681R_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484K_(—) 28676, D614G_P681R_D950N)28815 S_stab_PP(K986P_V987P_L452R_E484K_D614G_(—) 28677,P681R_R682del_R683del_A684del_R685del) 28816S_stab_PP(K986P_V987P_E154K_L452R_E484K_(—) 28678,D614G_P681R_R682del_R683del_A684del_(—) 28817 R685del_Q1071H)S_stab_PP(K986P_V987P_T19R_F157del_R158del_(—) 28679,L452R_T478K_E484K_D614G_P681R_R682del_(—) 28818R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_G142D_E156G_(—) 28680,F157del_R158del_L452R_T478K_E484K_D614G_(—) 28819P681R_R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28681,E156G_F157del_R158del_W258L_K417N_L452R_(—) 28820T478K_E484K_D614G_P681R_R682del_R683del_(—) A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_V70F_G142D_(—) 28682,E156G_F157del_R158del_A222V_K417N_L452R_(—) 28821T478K_E484K_D614G_P681R_R682del_R683del_(—) A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28683,E156G_F157del_R158del_L452R_T478K_E484K_(—) 28822D614G_P681R_R682del_R683del_A684del_R685del_(—) D950N)S_stab_PP(K986P_V987P_T19R_E156G_F157del_(—) 28684,R158del_L452R_T478K_E484K_D614G_P681R_(—) 28823R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_(—) 28685,Y145H_E156G_F157del_R158del_A222V_L452R_(—) 28824T478K_E484K_D614G_P681R_R682del_R683del_(—) A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484K_(—) 28686,D614G_P681R_R682del_R683del_A684del_(—) 28825 R685del_D950N)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28925,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28933L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)N440K_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_(—)N764K_D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v2)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28926,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28934L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v3)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28927,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28935L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_(—)Y505H_T547K_D614G_H655Y_N679K_P681H_A701V_(—)N764K_D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v4)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28928,T95I_G142D_V143del_Y144del_Y145del_N211del_(—) 28936L212I_ins214EPE_G339D_S371L_S373P_S375F_(—)G446S_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_(—)N764K_D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v5)

TABLE 2b Nucleic acid, preferably mRNA constructs suitable for acoronavirus vaccine Col A Col B Col C Col D Col E Col F Col GS_stab_PP(K986P_V987P_D614G) 24838, 24856 24874 24892 24910 24928 28827S_stab_PP(K986P_V987P_A222V_D614G) 24839, 24857 24875 24893 24911 2492928828 S_stab_PP(K986P_V987P_N439K_D614G) 24840, 24858 24876 24894 2491224930 28829 S_stab_PP(K986P_V987P_S477N_D614G) 24841, 24859 24877 2489524913 24931 28830 S_stab_PP(K986P_V987P_N501Y_D614G) 24842, 24860 2487824896 24914 24932 28831 S_stab_PP(K986P_V987P_H69del_V70del_D614G)24843, 24861 24879 24897 24915 24933 28832S_stab_PP(K986P_V987P_Y453F_D614G) 24844, 24862 24880 24898 24916 2493428833 S_stab_PP(K986P_V987P_D614G_I692V) 24845, 24863 24881 24899 2491724935 28834 S_stab_PP(K986P_V987P_D614G_M1229I) 24846, 24864 24882 2490024918 24936 28835 S_stab_PP(K986P_V987P_H69del_V70del_A222V_(—) 24847,24865 24883 24901 24919 24937 Y453F_S477N_D614G_I692V) 28836S_stab_PP(K986P_V987P_H69del_V70del_Y453F_(—) 24848, 24866 24884 2490224920 24938 D614G_I692V_M1229I) 28837S_stab_PP(K986P_V987P_H69del_V70del_Y144del_(—) 24849, 24867 24885 2490324921 24939 N501Y_A570D_D614G_P681H_T716I_S982A_D1118H) 28838S_stab_PP(K986P_V987P_K417N_E484K_N501Y_D614G) 24850, 24868 24886 2490424922 24940 28839 S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_(—)24851, 24869 24887 24905 24923 24941A243del_L244del_R246I_K417N_E484K_N501Y_D614G_A701V) 28840S_stab_PP(K986P_V987P_E484K_D614G) 24852, 24870 24888 24906 24924 2494228841 S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_(—) 24853, 2487124889 24907 24925 24943 K417T_E484K_N501Y_D614G_H655Y_T1027I) 28842S_stab_PP(K986P_V987P_S13I_W152C_L452R_D614G) 24854, 24872 24890 2490824926 24944 28843 S_stab_PP(K986P_V987P_H69del_V70del_Y144del_E484K_(—)27524, 27547 27570 27593 27616 27639N501Y_A570D_D614G_P681H_T716I_S982A_D1118H) 28844S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_(—) 27525, 27548 2757127594 27617 27640 A243del_L244del_K417N_E484K_N501Y_D614G_A701V) 28845S_stab_PP(K986P_V987P_Q52R_A67V_H69del_V70del_(—) 27526, 27549 2757227595 27618 27641 Y144del_E484K_D614G_Q677H_F888L) 28846S_stab_PP(K986P_V987P_A67V_H69del_V70del_Y144del_(—) 27527, 27550 2757327596 27619 27642 E484K_D614G_Q677H_F888L) 28847S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_(—) 27528, 27551 2757427597 27620 27643 K417T_E484K_N501Y_D614G_H655Y_T1027I_V1176F) 28848S_stab_PP(K986P_V987P_E484K_D614G_V1176F) 27529, 27552 27575 27598 2762127644 28849 S_stab_PP(K986P_V987P_L452R_D614G_P681R) 27530, 27553 2757627599 27622 27645 28850S_stab_PP(K986P_V987P_E154K_L452R_E484Q_D614G_(—) 27531, 27554 2757727600 27623 27646 P681R_Q1071H) 28851S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_(—) 27532, 27555 2757827601 27624 27647 T478K_D614G_P681R_D950N) 28852S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_(—) 27533, 27556 2757927602 27625 27648 Y248del_L249del_T250del_P251del_G252del_L452Q_(—)28853 F490S_D614G_T859N)S_stab_PP(K986P_V987P_H69del_V70del_N439K_D614G) 27534, 27557 2758027603 27626 27649 28854 S_stab_PP(K986P_V987P_L5F_T95I_D253G_E484K_(—)27535, 27558 27581 27604 27627 27650 D614G_A701V) 28855S_stab_PP(K986P_V987P_L5F_T95I_D253G_S477N_(—) 27536, 27559 27582 2760527628 27651 D614G_Q957R) 28856S_stab_PP(K986P_V987P_F157L_V367F_Q613H_P681R) 27537, 27560 27583 2760627629 27652 28857 S_stab_PP(K986P_V987P_S254F_D614G_P681R_G769V) 27538,27561 27584 27607 27630 27653 28858S_stab_PP(K986P_V987P_P26S_H69del_V70del_V126A_(—) 27539, 27562 2758527608 27631 27654 Y144del_L242del_A243del_L244del_H245Y_S477N_E484K_(—)28859 D614G_P681H_T1027I_D1118H)S_stab_PP(K986P_V987P_T95I_Y144T_Y145S_ins145N_(—) 27540, 27563 2758627609 27632 27655 R346K_E484K_N501Y_D614G_P681H_D950N) 28860S_stab_PP(K986P_V987P_ins214TDR_Q414K_N450K_(—) 27541, 27564 27587 2761027633 27656 D614G_T716I) 28861S_stab_PP(K986P_V987P_T478K_D614G_P681H_T732A) 27542, 27565 27588 2761127634 27657 28862 S_stab_PP(K986P_V987P_E484K_N501Y_D614G_P681H_(—)27543, 27566 27589 27612 27635 27658 E1092K_H1101Y_V1176F) 28863S_stab_PP(K986P_V987P_H66D_G142V_Y144del_Y145del_(—) 27544, 27567 2759027613 27636 27659 D215G_V483A_D614G_H655Y_G669S_Q949R_N1187D) 28864S_stab_PP(K986P_V987P_Y144del_L452R_T478K_P681R) 27545, 27568 2759127614 27637 27660 28865S_stab_PP(K986P_V987P_T19R_Y144del_Y145del_L452R_(—) 27546, 27569 2759227615 27638 27661 T478K_D614G_P681R) 28866S_stab_PP(K986P_V987P_P9L_C136F_Y144del_R190S_(—) 28687,D215G_L242del_A243del_Y449H_E484K_N501Y_D614G_(—) 28867H655Y_N679K_T716I_T859N)SS_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_(—) 28688,G142D_V143del_Y144del_Y145del_N211del_L212I_(—) 28868ins214EPE_G339D_S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_Y505H_(—)T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_N856K_(—) Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v1)S_stab_PP(K986P_V987P_A67V_H69del_V70del_(—) 28689,T95I_G142D_V143del_Y144del_Y145del_N211del_L212I_(—) 28869ins214EPE_G339D_S371L_S373P_S375F_K417N_N440K_(—)G446S_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_(—)Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_(—)N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v0)S_stab_PP(K986P_V987P_A67V_T95I_G339D_S371L_(—) 28690,S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—) 28870N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_(—)N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_B.1.1.529)S_stab_PP(K986P_V987P_T19I_L24del_P25del_P26del_(—) 28691,A27S_G142D_V213G_G339D_S371F_S373P_S375F_T376A_(—) 28871D405N_S477N_T478K_E484A_Q493R_Q498R_N501Y_Y505H_(—)D614G_H655Y_N679K_P681H_D796Y_Q954H_N969K); S_stab_PP(K986P_V987P_BA.2)S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_(—) 28692,Y248del_L249del_T250del_P251del_G252del_D253N_(—) 28872L452Q_F490S_D614G_T859N)S_stab_PP(K986P_V987P_T95I_Y144S_Y145N_R346K_(—) 28693,E484K_N501Y_D614G_P681H_D950N) 28873S_stab_PP(K986P_V987P_S12F_H69del_V70del_(—) 28694,W152R_R346S_L452R_D614G_Q677H_A899S) 28874S_stab_PP(K986P_V987P_I210T_N440K_E484K_D614G_(—) 28695,D936N_S939F_T1027I) 28875 S_stab_PP(K986P_V987P_W152L_E484K_D614G_G769V)28696, 28876 S_stab_PP(K986P_V987P_T20I_R357K_D614G) 28697, 28877S_stab_PP(K986P_V987P_T95I_Y144del_E484K_(—) 28698, D614G_P681H_D796H)28878 S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_(—) 28699,R158del_L452R_T478K_D614G_P681R_D950N) 28879S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_(—) 28700,F157del_R158del_W258L_K417N_L452R_T478K_D614G_(—) 28880 P681R_D950N)S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_(—) 28701,F157del_R158del_A222V_K417N_L452R_T478K_(—) 28881 D614G_P681R_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_(—) 28702,F157del_R158del_L452R_T478K_D614G_P681R_D950N) 28882S_stab_PP(K986P_V987P_T19R_E156G_F157del_(—) 28703,R158del_L452R_T478K_D614G_P681R_D950N) 28883S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_(—) 28704,E156G_F157del_R158del_A222V_L452R_T478K_D614G_(—) 28884 P681R_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484Q_D614G_(—) 28705, P681R_D950N)28885 S_stab_PP(K986P_V987P_L452R_D614G_P681R_R682del_(—) 28706,R683del_A684del_R685del) 28886S_stab_PP(K986P_V987P_E154K_L452R_E484Q_D614G_(—) 28707,P681R_R682del_R683del_A684del_R685del_Q1071H) 28887S_stab_PP(K986P_V987P_T19R_F157del_R158del_(—) 28708,L452R_T478K_D614G_P681R_R682del_R683del_A684del_(—) 28888 R685del_D950N)S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_(—) 28709,R158del_L452R_T478K_D614G_P681R_R682del_R683del_(—) 28889A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) 28710,R158del_W258L_K417N_L452R_T478K_D614G_P681R_R682del_(—) 28890R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_(—) 28711,R158del_A222V_K417N_L452R_T478K_D614G_P681R_R682del_(—) 28891R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) 28712,R158del_L452R_T478K_D614G_P681R_R682del_R683del_(—) 28892A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_(—) 28713,L452R_T478K_D614G_P681R_R682del_R683del_A684del_(—) 28893 R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_(—) 28714,F157del_R158del_A222V_L452R_T478K_D614G_P681R_(—) 28894R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484Q_D614G_P681R_(—) 28715,R682del_R683del_A684del_R685del_D950N) 28895S_stab_PP(K986P_V987P_L452R_E484K_D614G_P681R) 28716, 28896S_stab_PP(K986P_V987P_E154K_L452R_E484K_D614G_P681R_(—) 28717, Q1071H)28897 S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_(—) 28718,T478K_E484K_D614G_P681R_D950N) 28898S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_(—) 28719,L452R_T478K_E484K_D614G_P681R_D950N) 28899S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) 28720,R158del_W258L_K417N_L452R_T478K_E484K_D614G_P681R_D950N) 28900S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_(—) 28721,R158del_A222V_K417N_L452R_T478K_E484K_D614G_P681R_D950N) 28901S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) 28722,R158del_L452R_T478K_E484K_D614G_P681R_D950N) 28902S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_(—) 28723,L452R_T478K_E484K_D614G_P681R_D950N) 28903S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_(—) 28724,F157del_R158del_A222V_L452R_T478K_E484K_D614G_P681R_(—) 28904 D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484K_D614G_P681R_D950N) 28725, 28905S_stab_PP(K986P_V987P_L452R_E484K_D614G_P681R_R682del_(—) 28726,R683del_A684del_R685del) 28906S_stab_PP(K986P_V987P_E154K_L452R_E484K_D614G_P681R_(—) 28727,R682del_R683del_A684del_R685del_Q1071H) 28907S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_T478K_(—) 28728,E484K_D614G_P681R_R682del_R683del_A684del_R685del_D950N) 28908S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_R158del_(—) 28729,L452R_T478K_E484K_D614G_P681R_R682del_R683del_A684del_(—) 28909R685del_D950N) S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—)28730, R158del_W258L_K417N_L452R_T478K_E484K_D614G_P681R_R682del_(—)28910 R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_(—) 28731,R158del_A222V_K417N_L452R_T478K_E484K_D614G_P681R_R682del_(—) 28911R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) 28732,R158del_L452R_T478K_E484K_D614G_P681R_R682del_R683del_(—) 28912A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_(—) 28733,L452R_T478K_E484K_D614G_P681R_R682del_R683del_A684del_(—) 28913R685del_D950N) S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_(—)28734, F157del_R158del_A222V_L452R_T478K_E484K_D614G_P681R_(—) 28914R682del_R683del_A684del_R685del_D950N)S_stab_PP(K986P_V987P_T19R_L452R_E484K_D614G_P681R_(—) 28735,R682del_R683del_A684del_R685del_D950N) 28915S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) 28929,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) 28937S371L_S373P_S375F_N440K_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v2)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) 28930,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) 28938S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_N856K_(—)Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v3)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) 28931,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) 28939S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N701V_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v4)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) 28932,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) 28940S371L_S373P_S375F_G446S_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v5)

In certain embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of the SEQ ID NOs: 148, 149, 151, 162, 163, 165, 22792,22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812,22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835, 22837,22839, 23309-23368, 23370-23404, 23529-23588, 23590-23624, 24837-24944,27248-27907, 28638-28915, 28925-28940 or a fragment or variant of any ofthese sequences. In certain embodiments at least one, preferably alluracil nucleotides in said RNA sequences are replaced by pseudouridine(ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.

In certain embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of the SEQ ID NOs provided in Columns B-G of Table 2a orTable 2b or a fragment or variant of any of these sequences. In certainembodiments at least one, preferably all uracil nucleotides in said RNAsequences are replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In certain embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of the SEQ ID NOs: 27256, 27279, 27302, 27325, 27348, 27371,27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601,27624, 27647, 27688, 27729, 27770, 27811, 27852, 27893, 28650-28655,28699-28704, 28762, 28789-28794, 28852, 28879-28884 or a fragment orvariant of any of these sequences. In certain embodiments at least one,preferably all uracil nucleotides in said RNA sequences are replaced bypseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ)nucleotides.

In certain embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of the SEQ ID NOs: 27256, 27279, 27302, 27325, 27348, 27371,27394, 27417, 27440, 27463, 27486, 27509, 27532, 27555, 27578, 27601,27624, 27647, 27688, 27729, 27770, 27811, 27852, 27893, 28762, 28852 ora fragment or variant of any of these sequences. In certain embodimentsat least one, preferably all uracil nucleotides in said RNA sequencesare replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In certain embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of the SEQ ID NOs: 28639-28642, 28778-28781, 28688-28691,28868-28871, 28925-28940 or a fragment or variant of any of thesesequences. In certain embodiments at least one, preferably all uracilnucleotides in said RNA sequences are replaced by pseudouridine (ψ)nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802,22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408,23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776,28638-28686, 28777-28825, 28925-28928, 28933-28936 or a fragment orvariant of any of these sequences. Further information regardingrespective nucleic acid sequences is provided under <223> identifier ofthe respective SEQ ID NO in the sequence listing, and in Table 2 (see inparticular Column B-G).

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27394, 27417, 27486, 28762, 28650-28655,28789-28794 or a fragment or variant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27394, 27417, 27486, 28762 or a fragment orvariant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 28639-28642, 28778-28781, 28925-28928,28933-28936 or a fragment or variant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872,27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735,28867-28915, 28929-28932, 28937-28940 or a fragment or variant of any ofthese sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27532, 27555, 27624, 28852, 28699-28704,28879-28884 or a fragment or variant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27532, 27555, 27624, 28852 or a fragment orvariant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 28688-28691, 28868-28871, 28929-28932,28937-28940 or a fragment or variant of any of these sequences.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800, 22802,22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408,23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776,28638-28686, 28777-28825, 28925-28928, 28933-28936 or a fragment orvariant of any of these sequences, wherein at least one, preferably alluracil nucleotides in said RNA sequences are replaced by pseudouridine(ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27394, 27417, 27486, 28762, 28650-28655,28789-28794 or a fragment or variant of any of these sequences, whereinat least one, preferably all uracil nucleotides in said RNA sequencesare replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27394, 27417, 27486, 28762 or a fragment orvariant of any of these sequences, wherein at least one, preferably alluracil nucleotides in said RNA sequences are replaced by pseudouridine(ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 28639-28642, 28778-28781, 28925-28928,28933-28936 or a fragment or variant of any of these sequences, whereinat least one, preferably all uracil nucleotides in said RNA sequencesare replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872,27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735,28867-28915, 28929-28932, 28937-28940 or a fragment or variant of any ofthese sequences, wherein at least one, preferably all uracil nucleotidesin said RNA sequences are replaced by pseudouridine (ψ) nucleotidesand/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27532, 27555, 27624, 28852, 28699-28704,28879-28884 or a fragment or variant of any of these sequences, whereinat least one, preferably all uracil nucleotides in said RNA sequencesare replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 27532, 27555, 27624, 28852 or a fragment orvariant of any of these sequences, wherein at least one, preferably alluracil nucleotides in said RNA sequences are replaced by pseudouridine(ψ) nucleotides and/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA, preferably the mRNA, comprises orconsists of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 28688-28691, 28868-28871, 28929-28932,28937-28940 or a fragment or variant of any of these sequences, whereinat least one, preferably all uracil nucleotides in said RNA sequencesare replaced by pseudouridine (ψ) nucleotides and/orN1-methylpseudouridine (m1ψ) nucleotides.

In particular embodiments, the RNA of the invention may be preparedusing any method known in the art, including chemical synthesis such ase.g. solid phase RNA synthesis, as well as in vitro methods, such as RNAin vitro transcription reactions. Accordingly, in a preferredembodiment, the RNA is obtained by RNA in vitro transcription.

Accordingly, in preferred embodiments, the RNA of the invention ispreferably an in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “in vitro transcription”relate to a process wherein RNA is synthesized in a cell-free system (invitro). RNA may be obtained by DNA-dependent in vitro transcription ofan appropriate DNA template, which according to the present invention isa linearized plasmid DNA template or a PCR-amplified DNA template. Thepromoter for controlling RNA in vitro transcription can be any promoterfor any DNA-dependent RNA polymerase. Particular examples ofDNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNApolymerases. In a preferred embodiment of the present invention the DNAtemplate is linearized with a suitable restriction enzyme, before it issubjected to RNA in vitro transcription.

Reagents used in RNA in vitro transcription typically include: a DNAtemplate (linearized plasmid DNA or PCR product) with a promotersequence that has a high binding affinity for its respective RNApolymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6,or Syn5); ribonucleotide triphosphates (NTPs) for the four bases(adenine, cytosine, guanine and uracil); optionally, a cap analogue asdefined herein; optionally, further modified nucleotides as definedherein; a DNA-dependent RNA polymerase capable of binding to thepromoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNApolymerase); optionally, a ribonuclease (RNase) inhibitor to inactivateany potentially contaminating RNase; optionally, a pyrophosphatase todegrade pyrophosphate, which may inhibit RNA in vitro transcription;MgCl2, which supplies Mg2+ ions as a co-factor for the polymerase; abuffer (TRIS or HEPES) to maintain a suitable pH value, which can alsocontain antioxidants (e.g. DTT), and/or polyamines such as spermidine atoptimal concentrations, e.g. a buffer system comprising TRIS-Citrate asdisclosed in WO2017/109161.

In preferred embodiments, the cap1 structure of the RNA of the inventionis formed using co-transcriptional capping using tri-nucleotide capanalogues m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG. Apreferred cap1 analogue that may suitably be used in manufacturing thecoding RNA of the invention is m7G(5′)ppp(5′)(2′OMeA)pG.

In a particularly preferred embodiment, the cap1 structure of the RNA ofthe invention is formed using co-transcriptional capping usingtri-nucleotide cap analogue 3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG.

In other embodiments, a capO structure of the RNA of the invention isformed using co-transcriptional capping using cap analogue3′OMe-m7G(5′)ppp(5′)G.

In additional embodiments, the nucleotide mixture used in RNA in vitrotranscription may additionally comprise modified nucleotides as definedherein. In that context, preferred modified nucleotides may be selectedfrom pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine,and 5-methoxyuridine. In particular embodiments, uracil nucleotides inthe nucleotide mixture are replaced (either partially or completely) bypseudouridine (ψ) and/or N1-methylpseudouridine (m1ψ) to obtain amodified RNA.

In preferred embodiments, the nucleotide mixture used in RNA in vitrotranscription does not comprise modified nucleotides as defined herein.In preferred embodiments, the nucleotide mixture used in RNA in vitrotranscription does only comprise G, C, A and U nucleotides, and,optionally, a cap analog as defined herein.

In preferred embodiments, the nucleotide mixture (i.e. the fraction ofeach nucleotide in the mixture) used for RNA in vitro transcriptionreactions may be optimized for the given RNA sequence, preferably asdescribed in WO2015/188933, the entire contents of which are herebyincorporated by reference.

In this context the in vitro transcription has been performed in thepresence of a sequence optimized nucleotide mixture and optionally a capanalog, preferably wherein the sequence optimized nucleotide mixturedoes not comprise chemically modified nucleotides.

In this context a sequence-optimized nucleoside triphosphate (NTP) mixis a mixture of nucleoside triphosphates (NTPs) for use in an in vitrotranscription reaction of an RNA molecule of a given sequence comprisingthe four nucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, whereinthe fraction of each of the four nucleoside triphosphates (NTPs) in thesequence-optimized nucleoside triphosphate (NTP) mix corresponds to thefraction of the respective nucleotide in said RNA molecule. If aribonucleotide is not present in the RNA molecule, the correspondingnucleoside triphosphate is also not present in the sequence-optimizednucleoside triphosphate (NTP) mix.

In embodiments where more than one different RNA as defined herein haveto be produced, e.g. where 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent RNAs have to be produced (see second aspect), procedures asdescribed in WO2017/109134 may suitably be used.

In the context of RNA-based vaccine production, it may be required toprovide GMP-grade nucleic acids, e.g. a GMP grade RNA. GMP-grade RNA maybe produced using a manufacturing process approved by regulatoryauthorities. Accordingly, in a particularly preferred embodiment, RNAproduction is performed under current good manufacturing practice (GMP),implementing various quality control steps on DNA (template) and RNAlevel, preferably according to WO2016/180430. In preferred embodiments,the RNA of the invention is a GMP-grade RNA, particularly a GMP-grademRNA. Accordingly, an RNA for a vaccine is preferably a GMP grade RNA.

The obtained RNA products are preferably purified using PureMessenger®(CureVac, Tübingen, Germany; RP-HPLC according to WO2008/077592) and/ortangential flow filtration (as described in WO2016/193206) and/or oligod(T) purification (see WO2016/180430).

In a further preferred embodiment, the RNA is lyophilized (e.g.according to WO2016/165831 or WO2011/069586, the entire content of bothPCT applications are hereby incorporated by reference) to yield atemperature stable dried RNA (powder). The RNA may also be dried usingspray-drying or spray-freeze drying (e.g. according to WO2016/184575 orWO2016/184576) to yield a temperature stable RNA (powder) as definedherein. Accordingly, in the context of manufacturing and purifyingnucleic acid, in particular RNA, the disclosures of WO2017/109161,WO2015/188933, WO2016/180430, WO2008/077592, WO2016/193206,WO2016/165831, WO2011/069586, WO2016/184575, and WO2016/184576 areincorporated herewith by reference.

Accordingly, in preferred embodiments, the RNA is a dried RNA.

The term “dried RNA” as used herein has to be understood as RNA that hasbeen lyophilized, or spray-dried, or spray-freeze dried as defined aboveto obtain a temperature stable dried RNA (powder).

In preferred embodiments, the nucleic acid of the invention is apurified nucleic acid, particularly a purified RNA.

The term “purified nucleic acid” as used herein should be understood asnucleic acid which has a higher purity after certain purification stepsthan the starting material. Typical impurities that are essentially notpresent in purified nucleic acid comprise peptides or proteins,spermidine, BSA, abortive nucleic acid sequences, nucleic acidfragments, free nucleotides, bacterial impurities, or impurities derivedfrom purification procedures. Accordingly, it is desirable in thisregard for the “degree of nucleic acid purity” to be as close aspossible to 100%. It is also desirable for the degree of nucleic acidpurity that the amount of full-length nucleic acid is as close aspossible to 100%. Accordingly, “purified nucleic acid” as used hereinhas a degree of purity of more than 75%, 80%, 85%, very particularly90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% ormore. The degree of purity may for example be determined by ananalytical HPLC, wherein the percentages provided above correspond tothe ratio between the area of the peak for the target nucleic acid andthe total area of all peaks representing the by-products. Alternatively,the degree of purity may for example be determined by an analyticalagarose gel electrophoresis or capillary gel electrophoresis.

In preferred embodiments, the nucleic acid of the invention is apurified RNA.

The term “purified RNA” or “purified mRNA” as used herein should beunderstood as RNA which has a higher purity after certain purificationsteps (e.g. HPLC, TFF, Oligo d(T) purification, precipitation steps)than the starting material (e.g. in vitro transcribed RNA). Typicalimpurities that are essentially not present in purified RNA comprisepeptides or proteins (e.g. enzymes derived from DNA dependent RNA invitro transcription, e.g. RNA polymerases, RNases, pyrophosphatase,restriction endonuclease, DNase), spermidine, BSA, abortive RNAsequences, RNA fragments (short double stranded RNA fragments, abortivesequences etc.), free nucleotides (modified nucleotides, conventionalNTPs, cap analogue), template DNA fragments, buffer components (HEPES,TRIS, MgCl2) etc. Other potential impurities that may be derived frome.g. fermentation procedures comprise bacterial impurities (bioburden,bacterial DNA) or impurities derived from purification procedures(organic solvents etc.). Accordingly, it is desirable in this regard forthe “degree of RNA purity” to be as close as possible to 100%. It isalso desirable for the degree of RNA purity that the amount offull-length RNA transcripts is as close as possible to 100%.Accordingly, “purified RNA” as used herein has a degree of purity ofmore than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and most favorably 99% or more. The degree of purity mayfor example be determined by an analytical HPLC, wherein the percentagesprovided above correspond to the ratio between the area of the peak forthe target RNA and the total area of all peaks representing theby-products. Alternatively, the degree of purity may for example bedetermined by an analytical agarose gel electrophoresis or capillary gelelectrophoresis.

In particularly preferred embodiments, the RNA has been purified byRP-HPLC and/or TFF to remove double-stranded RNA, non-capped RNA and/orRNA fragments.

The formation of double stranded RNA as side products during e.g. RNA invitro transcription can lead to an induction of the innate immuneresponse, particularly IFNalpha which is the main factor of inducingfever in vaccinated subjects, which is of course an unwanted sideeffect. Current techniques for immunoblotting of dsRNA (via dot Blot,serological specific electron microscopy (SSEM) or ELISA for example)are used for detecting and sizing dsRNA species from a mixture ofnucleic acids.

Suitably, the RNA of the invention has been purified by RP-HPLC and/orTFF as described herein to reduce the amount of dsRNA.

Preferably, the RNA according to the invention is purified usingRP-HPLC, preferably using Reversed-Phase High pressure liquidchromatography (RP-HPLC) with a macroporous styrene/divinylbenzenecolumn (e.g. particle size 30 μm, pore size 4000 Å and additionallyusing a filter cassette with a cellulose-based membrane with a molecularweight cutoff of about 100 kDa.

In this context it is particularly preferred that the purified RNA hasbeen purified by RP-HPLC and/or TFF which results in about 5%, 10%, or20% less double stranded RNA side products as in RNA that has not beenpurified with RP-HPLC and/or TFF. Accordingly, the RNA of the inventioncomprises about 5%, 10%, or 20% less double stranded RNA side productsas an RNA that has not been purified with RP-HPLC and/or TFF.

Alternatively, the purified RNA that has been purified by RP-HPLC and/orTFF comprises about 5%, 10%, or 20% less double stranded RNA sideproducts as an RNA that has been purified with Oligo dT purification,precipitation, filtration and/or anion exchange chromatography.Accordingly, the RP-HPLC and/or TFF purified RNA of the inventioncomprises about 5%, 10%, or 20% less double stranded RNA side productsas an RNA that has been purified with Oligo dT purification,precipitation, filtration and/or AEX.

In embodiments, an automated device for performing RNA in vitrotranscription may be used to produce and purify the nucleic acid of theinvention. Such a device may also be used to produce the composition orthe vaccine (see aspects 2 and 3). Preferably, a device as described inWO2020/002598 (the entire content of which is hereby incorporated byrefernece), in particular, a device as described in claims 1 to 59and/or 68 to 76 of WO2020/002598 (and FIGS. 1-18) may suitably be used.

The methods described herein may preferably applied to a method ofproducing an RNA composition or vaccine as described in further detailbelow.

Composition, Pharmaceutical Composition:

A second aspect relates to a composition comprising at least one RNA ofthe first aspect.

Notably, embodiments relating to the composition of the second aspectmay likewise be read on and be understood as suitable embodiments of thevaccine of the third aspect. Also, embodiments relating to the vaccineof the third aspect may likewise be read on and be understood assuitable embodiments of the composition of the second aspect (comprisingat least one RNA of the first aspect). Furthermore, features andembodiments described in the context of the first aspect (the RNA of theinvention) have to be read on and have to be understood as suitableembodiments of the composition of the second aspect.

In preferred embodiments, the composition comprises at least one RNAaccording to the first aspect encoding at least one antigenic peptide orprotein that is or is derived from a SARS-CoV-2 spike protein, or animmunogenic fragment or immunogenic variant thereof.

In preferred embodiments, the composition comprises at least one RNAencoding at least one antigenic peptide or protein that is selected oris derived from a SARS-CoV-2 spike protein, or an immunogenic fragmentor immunogenic variant thereof according to the first aspect, whereinsaid composition is to be, preferably, administered intramuscularly orintradermal.

Preferably, intramuscular or intradermal administration of saidcomposition results in expression of the encoded SARS-CoV-2 spikeprotein construct in a subject. In preferred embodiments, administrationof the composition results in translation of the RNA and to a productionof the encoded SARS-CoV-2 spike protein in a subject.

Preferably, the composition of the second aspect is suitable for avaccine, in particular, suitable for a SARS-CoV-2 vaccine, preferably aSARS-CoV-2 vaccine against at least one of the following SARS-CoV-2isolates: C.1.2 (South Africa), B.1.1.529 (Omicron, South Africa)(including BA.1_v1, BA.1_v0, B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4,BA.1_v5), C.36.3 (Thailand), B.1.619 (Cameroon), R.1 (Kentucky, US),B.1.1.176 (Canada), AZ.3, AY.1 (India), AY.2 (India), AY.4 (India),AY.4.2 (Delta Plus, India), B.1.617.3 (India), B.1.351 (Beta, SouthAfrica), B.1.1.7 (Alpha, UK), P.1 (Gamma, Brazil), B.1.427/B.1.429(Epsilon, California, US), B.1.525 (Eta, Nigeria), B.1.258 (Czechrepublic), B.1.526 (Jota, New York, US), A.23.1 (Uganda), B.1.617.1(Kappa, India), B.1.617.2 (Delta, India), P.2 (Zeta, Brazil), C37.1(Lambda, Peru). P.3 (Theta, Philippines), and/or B.1.621 (Mu, Columbia).

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against B.1.351 (Beta, SouthAfrica).

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against B.1.617.2, AY.1,AY.2, AY.4 or AY.4.2.

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against B.1.617.2.

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against B.1.1.529,B.1.1.529.1/BA.1 (Omicron) and/or B.1.1.529.2/BA.2.

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, and/or BA.1_v5.

In particularly preferred embodiments, the composition of the secondaspect is suitable for a SARS-CoV-2 vaccine against B.1.1.529 andB.1.617.2.

In the context of the invention, a “composition” refers to any type ofcomposition in which the specified ingredients (e.g. RNA encoding atleast one antigenic peptide or protein that is selected or is derivedfrom SARS-CoV-2, e.g. in association with a lipid-based carrier) may beincorporated, optionally along with any further constituents, usuallywith at least one pharmaceutically acceptable carrier or excipient. Thecomposition may be a dry composition such as a powder or granules, or asolid unit such as a lyophilized form. Alternatively, the compositionmay be in liquid form, and each constituent may be independentlyincorporated in dissolved or dispersed (e.g. suspended or emulsified)form.

In a preferred embodiment of the second aspect, the compositioncomprises at least one RNA of the first aspect, and optionally, at leastone pharmaceutically acceptable carrier or excipient.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein preferably includes the liquid ornon-liquid basis of the composition for administration. If thecomposition is provided in liquid form, the carrier may be water, e.g.pyrogen-free water; isotonic saline or buffered (aqueous) solutions,e.g. phosphate, citrate etc. buffered solutions. Water or preferably abuffer, more preferably an aqueous buffer, may be used, containing asodium salt, preferably at least 50 mM of a sodium salt, a calcium salt,preferably at least 0.01 mM of a calcium salt, and optionally apotassium salt, preferably at least 3 mM of a potassium salt. Accordingto preferred embodiments, the sodium, calcium and, optionally, potassiumsalts may occur in the form of their halogenides, e.g. chlorides,iodides, or bromides, in the form of their hydroxides, carbonates,hydrogen carbonates, or sulfates, etc. Examples of sodium salts includeNaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optionalpotassium salts include KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examplesof calcium salts include CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂.

Furthermore, organic anions of the aforementioned cations may be in thebuffer. Accordingly, in embodiments, the nucleic acid composition maycomprise pharmaceutically acceptable carriers or excipients using one ormore pharmaceutically acceptable carriers or excipients to e.g. increasestability, increase cell transfection, permit the sustained or delayed,increase the translation of encoded coronavirus protein in vivo, and/oralter the release profile of encoded coronavirus protein in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients of the present inventioncan include, without limitation, lipidoids, liposomes, lipidnanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides,proteins, cells transfected with polynucleotides, hyaluronidase,nanoparticle mimics and combinations thereof. In embodiments, one ormore compatible solid or liquid fillers or diluents or encapsulatingcompounds may be used as well, which are suitable for administration toa subject. The term “compatible” as used herein means that theconstituents of the composition are capable of being mixed with the atleast one nucleic acid and, optionally, a plurality of nucleic acids ofthe composition, in such a manner that no interaction occurs, whichwould substantially reduce the biological activity or the pharmaceuticaleffectiveness of the composition under typical use conditions (e.g.,intramuscular or intradermal administration). Pharmaceuticallyacceptable carriers or excipients must have sufficiently high purity andsufficiently low toxicity to make them suitable for administration to asubject to be treated. Compounds which may be used as pharmaceuticallyacceptable carriers or excipients may be sugars, such as, for example,lactose, glucose, trehalose, mannose, and sucrose; starches, such as,for example, corn starch or potato starch; dextrose; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin;tallow; solid glidants, such as, for example, stearic acid, magnesiumstearate; calcium sulfate; vegetable oils, such as, for example,groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oilfrom theobroma; polyols, such as, for example, polypropylene glycol,glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

The at least one pharmaceutically acceptable carrier or excipient of thecomposition may preferably be selected to be suitable for intramuscularor intradermal delivery/administration of said composition. Accordingly,the composition is preferably a pharmaceutical composition, suitably acomposition for intramuscular administration.

Subjects to which administration of the compositions, preferably thepharmaceutical composition, is contemplated include, but are not limitedto, humans and/or other primates; mammals, including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice,and/or rats; and/or birds, including commercially relevant birds such aspoultry, chickens, ducks, geese, and/or turkeys.

Pharmaceutical compositions of the present invention may suitably besterile and/or pyrogen-free.

Multivalent Compositions of the Invention:

In embodiments, the composition (e.g. multivalent composition) asdefined herein may comprise a plurality or at least more than one of theRNA species as defined in the context of the first aspect of theinvention. Preferably, the composition as defined herein may comprise 2,3, 4, 5, 6, 7, 8, 9, or 10 different RNA species each defined in thecontext of the first aspect.

In embodiments, the composition (e.g. multivalent composition) comprisesat least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNA speciesas defined in the context of the first aspect, each encoding at leastone different SARS-CoV-2 spike protein (as defined in the context of thefirst aspect).

In this context it is further preferred that the different SARS-CoV-2spike proteins or prefusion stabilized spike proteins have amino acidchanges in the spike protein comprising:

K986, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,and L981F;

K986P, V987P, A67V, T95I, G339D, S371L, S373P, S375F, S477N, T478K,E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,P681H, D796Y, N856K, Q954H, N969K, and L981F;

K986P, V987P, T19I, L24del, P25del, P26del, A27S, G142D, V213G, G339D,S371F, S373P, S375F, T376A, D405N, S477N, T478K, E484A, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, D796Y, Q954H, and N969K;

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, N440K,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, D796Y, N856K, Q954H, N969K, and L981F;

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, A701V, N764K, D796Y, N856K, Q954H, N969K, and L981F;

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F;

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V;

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y, andT1027I;

E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, K417T, D614G, H655Y,T1027I, and V1176F;

L452R, P681R, and D614G;

L452R, E484Q, P681R, E154K, D614G, and Q1071H; or

L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.

In this context it is even more preferred that the different SARS-CoV-2spike proteins or prefusion stabilized spike proteins have amino acidchanges in the spike protein comprising:

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, R246I,K417N, D614G, and A701V; or

E484K, N501Y, L18F, D80A, D215G, L242del, A243del, L244del, K417N,D614G, and A701V.

In this context it is even more preferred that the different SARS-CoV-2spike proteins or prefusion stabilized spike proteins have amino acidchanges in the spike protein comprising:

-   -   at least one SARS-CoV-2 spike protein or prefusion stabilized        spike protein having the following amino acid changes in the        spike protein:

K986P, V987P, A67V, H69del, V70del, T95I, G142D, V143del, Y144del,Y145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F; and

-   -   at least one SARS-CoV-2 spike protein or prefusion stabilized        spike protein having the following amino acid changes in the        spike protein:    -   L452R, E484Q, P681R, E154K, D614G, and Q1071H; or    -   L452R, P681R, T19R, F157del, R158del, T478K, D614G, and D950N.    -   In preferred embodiments, the composition (e.g. multivalent        composition) comprises 2, 3, 4 or 5 RNA species, wherein said        RNA species comprise or consist of a nucleic acid sequence which        is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence selected from the group consisting of SEQ        ID NOs: 22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806,        22808, 22810, 22812, 23529-23534, 27386-27408, 23535-23552,        27409-27431, 23590-23606, 27478-27500, 28736-28776, 28638-28686,        28777-28825, 28925-28928, 28933-28936 and, optionally, at least        one pharmaceutically acceptable carrier or excipient, wherein        each of the 2, 3, 4 or 5 nucleic acid species encode a different        SARS-CoV-2 spike protein.

In preferred embodiments, the composition (e.g. multivalent composition)comprises 2, 3, 4 or 5 RNA species, wherein said RNA species comprise orconsist of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872,27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735,28867-28915, 28937-28940 and, optionally, at least one pharmaceuticallyacceptable carrier or excipient, wherein each of the 2, 3, 4 or 5nucleic acid species encode a different SARS-CoV-2 spike protein.

In the following, particularly preferred embodiments of a multivalentcomposition are provided.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNO: 10, wherein the multivalent composition additionally comprises atleast 2, 3, 4 further RNA species selected from

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540;        and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27093-27095,        28552-28558; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558;        and/or    -   vii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095; and/or    -   viii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOs: 27093-27095, 28552-28558, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540;        and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 10; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOs: 27095, 28552-28558, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540;        and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 10; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOs: 27095, wherein the multivalent composition additionally comprisesat least 2, 3, 4 further RNA species selected from

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540;        and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 10; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOs: 22960-22961, 28540, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 10; and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27093-27095,        28552-28558; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558;        and/or    -   vii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095; and/or    -   viii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising a coding sequence encoding an amino acid sequencebeing identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ IDNOs: 28541-28544, 28917-28920, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   -   i) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 27108-27109; and/or    -   ii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 10; and/or    -   iii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27093-27095,        28552-28558; and/or    -   iv) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27096, 28545; and/or    -   v) one RNA species comprises a coding sequence encoding an amino        acid sequence being identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to any one of SEQ ID NOs: 22959; and/or    -   vi) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095, 28552-28558;        and/or    -   vii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 27095; and/or    -   viii) one RNA species comprises a coding sequence encoding an        amino acid sequence being identical or at least 70%, 80%, 85%,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        or 99% identical to any one of SEQ ID NOs: 22960-22961, 28540.

In preferred embodiments, the composition, preferably the multivalentcomposition is suitable for a vaccine against C.1.2 (South Africa),B.1.1.529 (Omicron, South Africa) (including BA.1_v1, BA.1_v0,B.1.1.529, BA.2, BA.1_v2, BA.1_v3, BA.1_v4, BA.1_v5), C.36.3 (Thailand),B.1.619 (Cameroon), R.1 (Kentucky, US), B.1.1.176 (Canada), AZ.3, AY.1(India), AY.2 (India), AY.4 (India), AY.4.2 (Delta Plus, India),B.1.617.3 (India), B.1.351 (Beta, South Africa), B.1.1.7 (Alpha, UK),P.1 (Gamma, Brazil), B.1.427/B.1.429 (Epsilon, California, US), B.1.525(Eta, Nigeria), B.1.258 (Czech republic), B.1.526 (Jota, New York, US),A.23.1 (Uganda), B.1.617.1 (Kappa, India), B.1.617.2 (Delta, India), P.2(Zeta, Brazil), C37.1 (Lambda, Peru). P.3 (Theta, Philippines), and/orB.1.621 (Mu, Columbia).

In embodiments, the RNA as comprised in the composition is provided inan amount of about 100 ng to about 500 ug, in an amount of about 1 ug toabout 200 ug, in an amount of about 1 ug to about 100 ug, in an amountof about 5 ug to about 100 ug, preferably in an amount of about bug toabout 50 ug, specifically, in an amount of about 1 ug, 2 ug, 3 ug, 4 ug,5 ug, 6 ug, 7 ug, 8 ug, 9 ug, 10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug,20 ug, 25 ug, 30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70ug, 75 ug, 80 ug, 85 ug, 90 ug, 95 ug or 100 ug.

In case the composition comprises a plurality or at least more than oneof the RNA species as defined herein (multivalent composition), theamount of RNA for each RNA species is provided in an amount of about 100ng to about 500 ug, in an amount of about 1 ug to about 200 ug, in anamount of about 1 ug to about 100 ug, in an amount of about 5 ug toabout 100 ug, preferably in an amount of about bug to about 50 ug,specifically, in an amount of about 1 ug, 2 ug, 3 ug, 4 ug, 5 ug, 6 ug,7 ug, 8 ug, 9 ug, lOug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 20 ug, 25 ug,30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80ug, 85 ug, 90 ug, 95 ug or 100 ug.

In some embodiments, the amount of RNA for each RNA species isessentially equal in mass. In other embodiments, the amount of RNA foreach RNA species is selected to be equimolar.

Complexation:

In a preferred embodiment of the second aspect, the at least one RNA,preferably the at least one mRNA, is complexed or associated withfurther compound to obtain a complexed formulated composition. Acomplexed formulation may have the function of a transfection agent. Acomplexed formulated composition may also have the function ofprotecting the RNA and/or mRNA from degradation.

In a preferred embodiment of the second aspect, the at least one RNA,preferably the at least one mRNA, and optionally the at least onefurther RNA, is complexed or associated with, or at least partiallycomplexed or partially associated with one or more cationic orpolycationic compound, preferably cationic or polycationic polymer,cationic or polycationic polysaccharide, cationic or polycationic lipid,cationic or polycationic protein, cationic or polycationic peptide, orany combinations thereof.

The term “cationic or polycationic compound” as used herein will berecognized and understood by the person of ordinary skill in the art,and are for example intended to refer to a charged molecule, which ispositively charged at a pH value ranging from about 1 to 9, at a pHvalue ranging from about 3 to 8, at a pH value ranging from about 4 to8, at a pH value ranging from about 5 to 8, more preferably at a pHvalue ranging from about 6 to 8, even more preferably at a pH valueranging from about 7 to 8, most preferably at a physiological pH, e.g.ranging from about 7.2 to about 7.5. Accordingly, a cationic component,e.g. a cationic peptide, cationic protein, cationic polymer, cationicpolysaccharide, cationic lipid may be any positively charged compound orpolymer which is positively charged under physiological conditions. A“cationic or polycationic peptide or protein” may contain at least onepositively charged amino acid, or more than one positively charged aminoacid, e.g. selected from Arg, His, Lys or Orn. Accordingly,“polycationic” components are also within the scope exhibiting more thanone positive charge under the given conditions.

The cationic or polycationic compounds, being particularly preferred inthis context may be selected from the following list of cationic orpolycationic peptides or proteins of fragments thereof: protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides,pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB,SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably,the nucleic acid (e.g. DNA or RNA), e.g. the coding RNA, preferably themRNA, is complexed with one or more polycations, preferably withprotamine or oligofectamine, most preferably with protamine.

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene etc.; cationic lipids,e.g. DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9,oligofectamine; or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP etc., modified acrylates, suchas pDMAEMA etc., modified amidoamines such as pAMAM etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI, poly(propyleneimine), etc., polyallylamine,sugar backbone based polymers, such as cyclodextrin based polymers,dextran based polymers, etc., silan backbone based polymers, such asPMOXA-PDMS copolymers, etc., blockpolymers consisting of a combinationof one or more cationic blocks (e.g. selected from a cationic polymer asmentioned above) and of one or more hydrophilic or hydrophobic blocks(e.g. polyethyleneglycole); etc.

According to various embodiments, the composition of the presentinvention comprises at least one RNA, preferably at least one mRNA asdefined in the context of the first aspect, and a polymeric carrier.

The term “polymeric carrier” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and are e.g.intended to refer to a compound that facilitates transport and/orcomplexation of another compound (e.g. cargo nucleic acid). A polymericcarrier is typically a carrier that is formed of a polymer. A polymericcarrier may be associated to its cargo (e.g. DNA, or RNA) by covalent ornon-covalent interaction. A polymer may be based on different subunits,such as a copolymer.

Suitable polymeric carriers in that context may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PEGylated PLL and polyethylenimine(PEI), dithiobis(succinimidylpropionate) (DSP),Dimethyl-3,3′-dithiobispropionimidate (DTBP), poly(ethylene imine)biscarbamate (PEIC), poly(L-lysine) (PLL), histidine modified PLL,poly(N-vinylpyrrolidone) (PVP), poly(propylenimine (PPI),poly(amidoamine) (PAMAM), poly(amido ethylenimine) (SS-PAEI),triehtylenetetramine (TETA), poly(ß-aminoester), poly(4-hydroxy-L-proineester) (PHP), poly(allylamine), poly(α-[4-aminobutyl]-L-glycolic acid(PAGA), Poly(D,L-lactic-co-glycolid acid (PLGA),Poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene)s (PPZ),poly(phosphoester)s (PPE), poly(phosphoramidate)s (PPA),poly(N-2-hydroxypropylmethacrylamide) (pHPMA),poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) PPE_EA), galactosylated chitosan, N-dodecylatedchitosan, histone, collagen and dextran-spermine. In one embodiment, thepolymer may be an inert polymer such as, but not limited to, PEG. In oneembodiment, the polymer may be a cationic polymer such as, but notlimited to, PEI, PLL, TETA, poly(allylamine),Poly(N-ethyl-4-vinylpyridinium bromide), pHPMA and pDMAEMA. In oneembodiment, the polymer may be a biodegradable PEI such as, but notlimited to, DSP, DTBP and PEIC. In one embodiment, the polymer may bebiodegradable such as, but not limited to, histine modified PLL,SS-PAEI, poly(ß-aminoester), PHP, PAGA, PLGA, PPZ, PPE, PPA and PPE-EA.

Encapsulation/Complexation in LNPs:

In preferred embodiments of the second aspect, the at least one RNA,preferably the at least one mRNA, and optionally the at least onefurther RNA, is complexed, encapsulated, partially encapsulated, orassociated with one or more lipids (e.g. cationic lipids and/or neutrallipids), thereby forming lipid-based carriers such as liposomes, lipidnanoparticles (LNPs), lipoplexes, and/or nanoliposomes.

The liposomes, lipid nanoparticles (LNPs), lipoplexes, and/ornanoliposomes—incorporated RNA may be completely or partially located inthe interior space of the liposomes, lipid nanoparticles (LNPs),lipoplexes, and/or nanoliposomes, within the lipid layer/membrane, orassociated with the exterior surface of the lipid layer/membrane.

The incorporation of RNA into liposomes/LNPs is also referred to hereinas “encapsulation” wherein the RNA is entirely contained within theinterior space of the liposomes, lipid nanoparticles (LNPs), lipoplexes,and/or nanoliposomes. The purpose of incorporating RNA into liposomes,lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes is toprotect the RNA from an environment which may contain enzymes orchemicals or conditions that degrade nucleic acid and/or systems orreceptors that cause the rapid excretion of the nucleic acid. Moreover,incorporating RNA into liposomes, lipid nanoparticles (LNPs),lipoplexes, and/or nanoliposomes may promote the uptake of the RNA, andhence, may enhance the therapeutic effect of the RNA encoding antigenicSARS-CoV-2 spike proteins. Accordingly, incorporating the at least oneRNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/ornanoliposomes may be particularly suitable for a SARS-CoV-2 vaccine,e.g. for intramuscular and/or intradermal administration.

In this context, the terms “complexed” or “associated” refer to theessentially stable combination of RNA with one or more lipids intolarger complexes or assemblies without covalent binding.

The term “lipid nanoparticle”, also referred to as “LNP”, is notrestricted to any particular morphology, and include any morphologygenerated when a cationic lipid and optionally one or more furtherlipids are combined, e.g. in an aqueous environment and/or in thepresence of a an RNA. For example, a liposome, a lipid complex, alipoplex and the like are within the scope of a lipid nanoparticle(LNP).

Liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 nm and 500 nm in diameter.

LNPs of the invention are suitably characterized as microscopic vesicleshaving an interior aqua space sequestered from an outer medium by amembrane of one or more bilayers. Bilayer membranes of LNPs aretypically formed by amphiphilic molecules, such as lipids of syntheticor natural origin that comprise spatially separated hydrophilic andhydrophobic domains. Bilayer membranes of the liposomes can also beformed by amphophilic polymers and surfactants (e.g., polymerosomes,niosomes, etc.). In the context of the present invention, an LNPtypically serves to transport the at least one RNA to a target tissue.

Accordingly, in preferred embodiments of the second aspect, the at leastone RNA is complexed with one or more lipids thereby forming lipidnanoparticles (LNP). Preferably, said LNP is particularly suitable forintramuscular and/or intradermal administration. LNPs typically comprisea cationic lipid and one or more excipients selected from neutrallipids, charged lipids, steroids and polymer conjugated lipids (e.g.PEGylated lipid). The at least one RNA may be encapsulated in the lipidportion of the LNP or an aqueous space enveloped by some or the entirelipid portion of the LNP. The RNA or a portion thereof may also beassociated and complexed with the LNP. An LNP may comprise any lipidcapable of forming a particle to which the RNA are attached, or in whichthe one or more RNA species are encapsulated. Preferably, the LNPcomprising RNA comprises one or more cationic lipids, and one or morestabilizing lipids. Stabilizing lipids include neutral lipids andPEGylated lipids.

Preferably, the LNP of the invention comprises

(i) at least one cationic lipid;

(ii) at least one neutral lipid;

(iii) at least one steroid or steroid analogue, preferably cholesterol;and

(iv) at least one polymer conjugated lipid, preferably a PEG-lipid;

wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid,5-25% neutral lipid, 25-55% sterol, and 0.5-15% polymer conjugatedlipid.

The cationic lipid of an LNP may be cationisable, i.e. it becomesprotonated as the pH is lowered below the pK of the ionizable group ofthe lipid, but is progressively more neutral at higher pH values. At pHvalues below the pK, the lipid is then able to associate with negativelycharged nucleic acids. In certain embodiments, the cationic lipidcomprises a zwitterionic lipid that assumes a positive charge on pHdecrease.

Such lipids include, but are not limited to, DSDMA,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known asN-(2,3-dioleoyloxy)propyI)-N,N,N-trimethylammonium chloride and1,2-Dioleyloxy-3-trimethylaminopropane chloride salt),N-(1-(2,3-dioleyloxy)propyI)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk,1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5,1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),ICE (Imidazol-based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA,DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) HGT4003,1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate(MC3), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA),3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(MC3 Ether),4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine(MC4 Ether), LIPOFECTIN® (commercially available cationic liposomescomprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), fromGIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially availablecationic liposomes comprisingN-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.) or any combination of any of the foregoing.Further suitable cationic lipids for use in the compositions and methodsof the invention include those described in international patentpublications WO2010/053572 (and particularly, CI 2-200 described atparagraph [00225]) and WO2012/170930, both of which are incorporatedherein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (seeUS2015/0140070A1).

In embodiments, the cationic lipid may be an amino lipid.

Representative amino lipids include, but are not limited to,1,2-dilinoleyoxy-3-(dimethylamino)acetoxpropane (DLin-DAC),1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxpropane (DLin-EG-DMA), and2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA);dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3(US20100324120).

In embodiments, the cationic lipid may an aminoalcohol lipidoid.

Aminoalcohol lipidoids which may be used in the present invention may beprepared by the methods described in U.S. Pat. No. 8,450,298, hereinincorporated by reference in its entirety. Suitable (ionizable) lipidscan also be the compounds as disclosed in Tables 1, 2 and 3 and asdefined in claims 1-24 of WO2017/075531A1, hereby incorporated byreference.

In another embodiment, suitable lipids can also be the compounds asdisclosed in WO2015/074085A1 (i.e. ATX-001 to ATX-032 or the compoundsas specified in claims 1-26), U.S. Appl. No. 61/905,724 and Ser. No.15/614,499 or U.S. Pat. Nos. 9,593,077 and 9,567,296 hereby incorporatedby reference in their entirety.

In other embodiments, suitable cationic lipids can also be the compoundsas disclosed in WO2017/117530A1 (i.e. lipids 13, 14, 15, 16, 17, 18, 19,20, or the compounds as specified in the claims), hereby incorporated byreference in its entirety.

In preferred embodiments, ionizable or cationic lipids may also beselected from the lipids disclosed in WO2018/078053A1 (i.e. lipidsderived from formula I, II, and III of WO2018/078053A1, or lipids asspecified in claims 1 to 12 of WO2018/078053A1), the disclosure ofWO2018/078053A1 hereby incorporated by reference in its entirety. Inthat context, lipids disclosed in Table 7 of WO2018/078053A1 (e.g.lipids derived from formula I-1 to I-41) and lipids disclosed in Table 8of WO2018/078053A1 (e.g. lipids derived from formula II-1 to II-36) maybe suitably used in the context of the invention. Accordingly, formulaI-1 to formula I-41 and formula II-1 to formula II-36 ofWO2018/078053A1, and the specific disclosure relating thereto, areherewith incorporated by reference.

In preferred embodiments, cationic lipids may be derived from formulaIII of published PCT patent application WO2018/078053A1. Accordingly,formula III of WO2018/078053A1, and the specific disclosure relatingthereto, are herewith incorporated by reference.

In particularly preferred embodiments, the at least one RNA, preferablythe at least one mRNA of the composition is complexed with one or morelipids thereby forming LNPs, wherein the cationic lipid of the LNP isselected from structures III-1 to III-36 of Table 9 of published PCTpatent application WO2018/078053A1. Accordingly, formula III-1 to III-36of WO2018/078053A1, and the specific disclosure relating thereto, areherewith incorporated by reference.

In particularly preferred embodiment of the second aspect, the at leastone RNA, preferably the at least one mRNA is complexed with one or morelipids thereby forming LNPs, wherein the LNPs comprises a cationic lipidaccording to formula III-3:

The lipid of formula III-3 as suitably used herein has the chemical term((4-hydroxybutypazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),also referred to as ALC-0315.

In certain embodiments, the cationic lipid as defined herein, morepreferably cationic lipid compound III-3, is present in the LNP in anamount from about 30 to about 95 mole percent, relative to the totallipid content of the LNP. If more than one cationic lipid isincorporated within the LNP, such percentages apply to the combinedcationic lipids. In embodiments, the cationic lipid is present in theLNP in an amount from about 30 to about 70 mole percent. In oneembodiment, the cationic lipid is present in the LNP in an amount fromabout 40 to about 60 mole percent, such as about 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 molepercent, respectively. In embodiments, the cationic lipid is present inthe LNP in an amount from about 47 to about 48 mole percent, such asabout 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0mole percent, respectively, wherein 47.7 mole percent are particularlypreferred.

In some embodiments, the cationic lipid is present in a ratio of fromabout 20 mol % to about 70 or 75 mol % or from about 45 to about 65 mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol % ofthe total lipid present in the LNP. In further embodiments, the LNPscomprise from about 25% to about 75% on a molar basis of cationic lipid,e.g., from about 20 to about 70%, from about 35 to about 65%, from about45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about40% on a molar basis (based upon 100% total moles of lipid in the lipidnanoparticle). In some embodiments, the ratio of cationic lipid to RNAis from about 3 to about 15, such as from about 5 to about 13 or fromabout 7 to about 11.

Other suitable (cationic or ionizable) lipids are disclosed inWO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406,WO2010/088537, WO2010/129709, WO2011/153493, WO 2013/063468,US2011/0256175, US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601,WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620,WO2017/099823, WO2012/040184, WO2011/153120, WO2011/149733,WO2011/090965, WO2011/043913, WO2011/022460, WO2012/061259,WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865,WO2008/103276, WO2013/086373, WO2013/086354, U.S. Pat. Nos. 7,893,302,7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent PublicationNo. US2010/0036115, US2012/0202871, US2013/0064894, US2013/0129785,US2013/0150625, US2013/0178541, US2013/0225836, US2014/0039032 andWO2017/112865. In that context, the disclosures of WO2009/086558,WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537,WO2010/129709, WO2011/153493, WO 2013/063468, US2011/0256175,US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601, WO2016/118724,WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823,WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965,WO2011/043913, WO2011/022460, WO2012/061259, WO2012/054365,WO2012/044638, WO2010/080724, WO2010/21865, WO2008/103276,WO2013/086373, WO2013/086354, U.S. Pat. Nos. 7,893,302, 7,404,969,8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No.US2010/0036115, US2012/0202871, US2013/0064894, US2013/0129785,US2013/0150625, US2013/0178541, US2013/0225836 and US2014/0039032 andWO2017/112865 specifically relating to (cationic) lipids suitable forLNPs are incorporated herewith by reference.

In embodiments, amino or cationic lipids as defined herein have at leastone protonatable or deprotonatable group, such that the lipid ispositively charged at a pH at or below physiological pH (e.g. pH 7.4),and neutral at a second pH, preferably at or above physiological pH. Itwill, of course, be understood that the addition or removal of protonsas a function of pH is an equilibrium process, and that the reference toa charged or a neutral lipid refers to the nature of the predominantspecies and does not require that all of lipids have to be present inthe charged or neutral form.

Lipids having more than one protonatable or deprotonatable group, orwhich are zwitterionic, are not excluded and may likewise suitable inthe context of the present invention. In some embodiments, theprotonatable lipids have a pKa of the protonatable group in the range ofabout 4 to about 11, e.g., a pKa of about 5 to about 7.

LNPs can comprise two or more (different) cationic lipids as definedherein. Cationic lipids may be selected to contribute to differentadvantageous properties. For example, cationic lipids that differ inproperties such as amine pKa, chemical stability, half-life incirculation, half-life in tissue, net accumulation in tissue, ortoxicity can be used in the LNP. In particular, the cationic lipids canbe chosen so that the properties of the mixed-LNP are more desirablethan the properties of a single-LNP of individual lipids.

The amount of the permanently cationic lipid or lipidoid may be selectedtaking the amount of the nucleic acid cargo into account. In oneembodiment, these amounts are selected such as to result in an N/P ratioof the nanoparticle(s) or of the composition in the range from about 0.1to about 20. In this context, the N/P ratio is defined as the mole ratioof the nitrogen atoms (“N”) of the basic nitrogen-containing groups ofthe lipid or lipidoid to the phosphate groups (“P”) of the nucleic acidwhich is used as cargo. The N/P ratio may be calculated on the basisthat, for example, 1 ug RNA typically contains about 3 nmol phosphateresidues, provided that the RNA exhibits a statistical distribution ofbases. The “N”-value of the lipid or lipidoid may be calculated on thebasis of its molecular weight and the relative content of permanentlycationic and—if present—cationisable groups.

In vivo characteristics and behavior of LNPs can be modified by additionof a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to theLNP surface to confer steric stabilization. Furthermore, LNPs can beused for specific targeting by attaching ligands (e.g. antibodies,peptides, and carbohydrates) to its surface or to the terminal end ofthe attached PEG chains (e.g. via PEGylated lipids or PEGylatedcholesterol).

In some embodiments, the LNPs comprise a polymer conjugated lipid. Theterm “polymer conjugated lipid” refers to a molecule comprising both alipid portion and a polymer portion. An example of a polymer conjugatedlipid is a PEGylated lipid. The term “PEGylated lipid” refers to amolecule comprising both a lipid portion and a polyethylene glycolportion. PEGylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG)and the like.

A polymer conjugated lipid as defined herein, e.g. a PEG-lipid, mayserve as an aggregation reducing lipid.

In certain embodiments, the LNP comprises a stabilizing-lipid which is apolyethylene glycol-lipid (PEGylated lipid). Suitable polyethyleneglycol-lipids include PEG-modified phosphatidylethanolamine,PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14or PEG-CerC20), PEG-modified dialkylamines, PEG-modifieddiacylglycerols, PEG-modified dialkylglycerols. Representativepolyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine(PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid isPEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid isPEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylateddiacylglycerol (PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), aPEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

In preferred embodiments, the PEGylated lipid is preferably derived fromformula (IV) of published PCT patent application WO2018/078053A1.Accordingly, PEGylated lipids derived from formula (IV) of published PCTpatent application WO2018/078053A1, and the respective disclosurerelating thereto, are herewith incorporated by reference.

In a particularly preferred embodiments, the at least one RNA of thecomposition is complexed with one or more lipids thereby forming LNPs,wherein the LNP comprises a PEGylated lipid, wherein the PEG lipid ispreferably derived from formula (IVa) of published PCT patentapplication WO2018/078053A1. Accordingly, PEGylated lipid derived fromformula (IVa) of published PCT patent application WO2018/078053A1, andthe respective disclosure relating thereto, is herewith incorporated byreference.

In a particularly preferred embodiment, the at least one RNA iscomplexed with one or more lipids thereby forming lipid nanoparticles(LNP), wherein the LNP comprises a PEGylated lipid/PEG lipid.Preferably, said PEG lipid is of formula (IVa):

wherein n has a mean value ranging from 30 to 60, such as about 30±2,32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2,56±2, 58±2, or 60±2. In a most preferred embodiment n is about 49. Infurther preferred aspects said PEG lipid is of formula (IVa) wherein nis an integer selected such that the average molecular weight of the PEGlipid is about 2000 g/mol to about 3000 g/mol or about 2300 g/mol toabout 2700 g/mol, even more preferably about 2500 g/mol.

The lipid of formula IVa as suitably used herein has the chemical term2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referredto as ALC-0159.

Further examples of PEG-lipids suitable in that context are provided inUS2015/0376115A1 and WO2015/199952, each of which is incorporated byreference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 molepercent of PEG or PEG-modified lipid, based on the total moles of lipidin the LNP. In further embodiments, LNPs comprise from about 0.1% toabout 20% of the PEG-modified lipid on a molar basis, e.g., about 0.5 toabout 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about3%, about 2,5%, about 2%, about 1.5%, about 1%, about 0.5%, or about0.3% on a molar basis (based on 100% total moles of lipids in the LNP).In preferred embodiments, LNPs comprise from about 1.0% to about 2.0% ofthe PEG-modified lipid on a molar basis, e.g., about 1.2 to about 1.9%,about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about1.8%, about 1.5 to about 1.8%, about 1.6 to about 1.8%, in particularabout 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%,most preferably 1.7% (based on 100% total moles of lipids in the LNP).In various embodiments, the molar ratio of the cationic lipid to thePEGylated lipid ranges from about 100:1 to about 25:1.

In preferred embodiments, the LNP comprises one or more additionallipids, which stabilize the formation of particles during theirformation or during the manufacturing process (e.g. neutral lipid and/orone or more steroid or steroid analogue).

In preferred embodiments of the second aspect, the at least one RNA iscomplexed with one or more lipids thereby forming lipid nanoparticles(LNP), wherein the LNP comprises one or more neutral lipid and/or one ormore steroid or steroid analogue.

Suitable stabilizing lipids include neutral lipids and anionic lipids.The term “neutral lipid” refers to any one of a number of lipid speciesthat exist in either an uncharged or neutral zwitterionic form atphysiological pH. Representative neutral lipids includediacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

In embodiments of the second aspect, the LNP comprises one or moreneutral lipids, wherein the neutral lipid is selected from the groupcomprising distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE), or mixtures thereof.

In some embodiments, the LNPs comprise a neutral lipid selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, themolar ratio of the cationic lipid to the neutral lipid ranges from about2:1 to about 8:1.

In preferred embodiments, the neutral lipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The molar ratio ofthe cationic lipid to DSPC may be in the range from about 2:1 to about8:1.

In preferred embodiments, the steroid is cholesterol. The molar ratio ofthe cationic lipid to cholesterol may be in the range from about 2:1 toabout 1:1. In some embodiments, the cholesterol may be PEGylated.

The sterol can be about 10 mol % to about 60 mol % or about 25 mol % toabout 40 mol % of the lipid particle. In one embodiment, the sterol isabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol % of thetotal lipid present in the lipid particle. In another embodiment, theLNPs include from about 5% to about 50% on a molar basis of the sterol,e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on amolar basis (based upon 100% total moles of lipid in the lipidnanoparticle).

Preferably, lipid nanoparticles (LNPs) comprise: (a) the at least oneRNA of the first aspect, (b) a cationic lipid, (c) an aggregationreducing agent (such as polyethylene glycol (PEG) lipid or PEG-modifiedlipid), (d) optionally a non-cationic lipid (such as a neutral lipid),and (e) optionally, a sterol.

In some embodiments, the cationic lipids (as defined above),non-cationic lipids (as defined above), cholesterol (as defined above),and/or PEG-modified lipids (as defined above) may be combined at variousrelative molar ratios. For example, the ratio of cationic lipid tonon-cationic lipid to cholesterol-based lipid to PEGylated lipid may bebetween about 30-60:20-35:20-30:1-15, or at a ratio of about 40:30:25:5,50:25:20:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3 or40:33:25:2, or at a ratio of about 50:25:20:5, 50:20:25:5, 50:27:20:340:30:20: 10,40:30:25:5 or 40:32:20:8, 40:32:25:3 or 40:33:25:2,respectively.

In some embodiments, the LNPs comprise a lipid of formula (III), the atleast one RNA as defined herein, a neutral lipid, a steroid and aPEGylated lipid. In preferred embodiments, the lipid of formula (III) islipid compound III-3 (ALC-0315), the neutral lipid is DSPC, the steroidis cholesterol, and the PEGylated lipid is the compound of formula (IVa)(ALC-0159).

In a preferred embodiment of the second aspect, the LNP consistsessentially of (i) at least one cationic lipid; (ii) a neutral lipid;(iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g. PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In particularly preferred embodiments, the at least RNA is complexedwith one or more lipids thereby forming lipid nanoparticles (LNP),wherein the LNP comprises

-   -   (i) at least one cationic lipid as defined herein, preferably a        lipid of formula (III), more preferably lipid III-3 (ALC-0315);    -   (ii) at least one neutral lipid as defined herein, preferably        1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);    -   (iii) at least one steroid or steroid analogue as defined        herein, preferably cholesterol; and    -   (iv) at least one PEG-lipid as defined herein, e.g. PEG-DMG or        PEG-cDMA, preferably a PEGylated lipid that is or is derived        from formula (IVa) (ALC-0159).

In particularly preferred embodiments, the at least one RNA is complexedwith one or more lipids thereby forming lipid nanoparticles (LNP),wherein the LNP comprises (i) to (iv) in a molar ratio of about 20-60%cationic lipid:5-25% neutral lipid:25-55% sterol; 0.5-15% PEG-lipid.

In one preferred embodiment, the lipid nanoparticle comprises: acationic lipid with formula (III) and/or PEG lipid with formula (IV),optionally a neutral lipid, preferably1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally asteroid, preferably cholesterol, wherein the molar ratio of the cationiclipid to DSPC is optionally in the range from about 2:1 to 8:1, whereinthe molar ratio of the cationic lipid to cholesterol is optionally inthe range from about 2:1 to 1.1.

In a particular preferred embodiment, the composition of the secondaspect comprising the at least one RNA, comprises lipid nanoparticles(LNPs), which have a molar ratio of approximately 50:10:38.5:1.5,preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e.proportion (mol %) of cationic lipid (preferably lipid III-3(ALC-0315)), DSPC, cholesterol and PEG-lipid (preferably PEG-lipid offormula (IVa) with n=49, even more preferably PEG-lipid of formula (IVa)with n=45 (ALC-0159)); solubilized in ethanol).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 24837-24854, 27524-27546,24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866,28687-28735, 28867-28915, 28929-28932, 28937-28940 formulated in lipidnanoparticles (LNPs), which have a molar ratio of approximately50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3 (ALC-0315),DSPC, cholesterol and PEG-lipid of formula (IVa) (with n=49 or with n=45(ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 27532, 27555, 27624, 28852,28699-28704, 28879-28884 formulated in lipid nanoparticles (LNPs), whichhave a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45 (ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 27532, 27555, 27624, 28852formulated in lipid nanoparticles (LNPs), which have a molar ratio ofapproximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or morepreferably 47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3(ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (with n=49or with n=45 (ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 28688-28691, 28868-28871,28929-28932, 28937-28940 formulated in lipid nanoparticles (LNPs), whichhave a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45 (ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800,22802, 22804, 22806, 22808, 22810, 22812, 23529-23534, 27386-27408,23535-23552, 27409-27431, 23590-23606, 27478-27500, 28736-28776,28638-28686, 28777-28825, 28925-28928, 28933-28936 formulated in lipidnanoparticles (LNPs), which have a molar ratio of approximately50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3 (ALC-0315),DSPC, cholesterol and PEG-lipid of formula (IVa) (with n=49 or with n=45(ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 27394, 27417, 27486, 28762,28650-28655, 28789-28794 formulated in lipid nanoparticles (LNPs), whichhave a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45 (ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 27394, 27417, 27486, 28762formulated in lipid nanoparticles (LNPs), which have a molar ratio ofapproximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or morepreferably 47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3(ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (with n=49or with n=45 (ALC-0159)).

Preferably, the composition of the second aspect comprises at least oneRNA, which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence of SEQ ID NOs: 28639-28642, 28778-28781,28925-28928, 28933-28936 formulated in lipid nanoparticles (LNPs), whichhave a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45 (ALC-0159)).

In embodimens where the composition is a multivalent composition asdefined above, the RNA species, preferably mRNA species of themultivalent composition may be formulated separately, e.g. may beformulated separately in liposomes or LNPs. Suitably, the RNA species ofthe multivalent composition are separately formulated in LNPs which havea molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45). Nucleic acid species formultivalent compositions are preferably selected as defined above (seesection “Multivalent compositions of the invention”).

In that context, the composition may comprise

-   -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NO: 149 formulated in lipid nanoparticles        (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 22792, 22794, 22796, 22798, 22800,        22802, 22804, 22806, 22808, 22810, 22812, 23529-23534,        27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500,        28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936        formulated in lipid nanoparticles (LNPs), which have a molar        ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 27394, 27417, 27486, 28762, 28650-28655,        28789-28794 formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 27394, 27417, 27486, 28762 formulated in        lipid nanoparticles (LNPs), which have a molar ratio of        approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or        more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic        lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of        formula (IVa) (with n=49 or with n=45 (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 28639-28642, 28778-28781, 28925-28928,        28933-28936 formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)).

In that context, the composition may comprise

-   -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NO: 24837 formulated in lipid nanoparticles        (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 24837-24854, 27524-27546, 24855-24872,        27547-27569, 24909-24926, 27616-27638, 28827-28866, 28687-28735,        28867-28915, 28929-28932, 28937-28940 formulated in lipid        nanoparticles (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 27532, 27555, 27624, 28852, 28699-28704,        28879-28884 formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 27532, 27555, 27624, 28852 formulated in        lipid nanoparticles (LNPs), which have a molar ratio of        approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or        more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic        lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of        formula (IVa) (with n=49 or with n=45 (ALC-0159)); and/or    -   at least one RNA, which is identical or at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid        sequence of SEQ ID NOs: 28688-28691, 28868-28871, 28929-28932,        28937-28940 formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)).

In embodiments where the composition is a multivalent composition asdefined above, the nucleic acid species (e.g. DNA or RNA), preferablyRNA species of the multivalent composition may be co-formulated,preferably co-formulated in liposomes or LNPs. Suitably, the RNA speciesof the multivalent composition are co-formulated in LNPs which have amolar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7or more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic lipid111-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (withn=49 or with n=45). Nucleic acid species for multivalent compositionsare preferably selected as defined above (see section “Multivalentcompositions of the invention”)

The total amount of RNA in the lipid nanoparticles may vary and isdefined depending on the e.g. nucleic acid to total lipid w/w ratio. Inone embodiment of the invention the nucleic acid, in particular the RNAto total lipid ratio is less than 0.06 w/w, preferably between 0.03 w/wand 0.04 w/w.

In some embodiments, the lipid nanoparticles (LNPs), which are composedof only three lipid components, namely imidazole cholesterol ester(ICE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K).

In one embodiment, the lipid nanoparticle of the composition comprises acationic lipid, a steroid; a neutral lipid; and a polymer conjugatedlipid, preferably a pegylated lipid. Preferably, the polymer conjugatedlipid is a pegylated lipid or PEG-lipid. In a specific embodiment, lipidnanoparticles comprise a cationic lipid resembled by the cationic lipidCOATSOME® SS-EC (former name: SS-33/4PE-15; NOF Corporation, Tokyo,Japan), in accordance with the following formula

As described further below, those lipid nanoparticles are termed “GN01”.

Furthermore, in a specific embodiment, the GN01 lipid nanoparticlescomprise a neutral lipid being resembled by the structure1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE):

Furthermore, in a specific embodiment, the GN01 lipid nanoparticlescomprise a polymer conjugated lipid, preferably a pegylated lipid, being1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) having the following structure:

As used in the art, “DMG-PEG 2000” is considered a mixture of1,2-DMG-PEG 2000 and 1,3-DMG-PEG 2000 in ˜97:3 ratio.

Accordingly, GN01 lipid nanoparticles (GN01-LNPs) according to one ofthe preferred embodiments comprise a SS-EC cationic lipid, neutral lipidDPhyPE, cholesterol, and the polymer conjugated lipid (pegylated lipid)1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG).

In a preferred embodiment, the GN01 LNPs comprise:

(a) cationic lipid SS-EC (former name: SS-33/4PE-15; NOF Corporation,Tokyo, Japan) at an amount of 45-65 mol %;

(b) cholesterol at an amount of 25-45 mol %;

(c) DPhyPE at an amount of 8-12 mol %; and

(d) PEG-DMG 2000 at an amount of 1-3 mol %;

each amount being relative to the total molar amount of all lipidicexcipients of the GN01 lipid nanoparticles.

In a further preferred embodiment, the GN01 lipid nanoparticles asdescribed herein comprises 59 mol % cationic lipid, 10 mol % neutrallipid, 29.3 mol % steroid and 1.7 mol % polymer conjugated lipid,preferably pegylated lipid. In a most preferred embodiment, the GN01lipid nanoparticles as described herein comprise 59 mol % cationic lipidSS-EC, 10 mol % DPhyPE, 29.3 mol % cholesterol and 1.7 mol % DMG-PEG2000.

The amount of the cationic lipid relative to that of the nucleic acid inthe GN01 lipid nanoparticle may also be expressed as a weight ratio(abbreviated f.e. “m/m”). For example, the GN01 lipid nanoparticlescomprise the at least one nucleic acid, preferably the at least one RNAat an amount such as to achieve a lipid to RNA weight ratio in the rangeof about 20 to about 60, or about 10 to about 50. In other embodiments,the ratio of cationic lipid to nucleic acid or RNA is from about 3 toabout 15, such as from about 5 to about 13, from about 4 to about 8 orfrom about 7 to about 11. In a very preferred embodiment of the presentinvention, the total lipid/RNA mass ratio is about 40 or 40, i.e. about40 or 40 times mass excess to ensure RNA encapsulation. Anotherpreferred RNA/lipid ratio is between about 1 and about 10, about 2 andabout 5, about 2 and about 4, or preferably about 3.

Further, the amount of the cationic lipid may be selected taking theamount of the nucleic acid cargo such as the RNA compound into account.In one embodiment, the N/P ratio can be in the range of about 1 to about50. In another embodiment, the range is about 1 to about 20, about 1 toabout 10, about 1 to about 5. In one preferred embodiment, these amountsare selected such as to result in an N/P ratio of the GN01 lipidnanoparticles or of the composition in the range from about 10 to about20. In a further very preferred embodiment, the N/P is 14 (i.e. 14 timesmol excess of positive charge to ensure nucleic acid encapsulation).

In a preferred embodiment, GN01 lipid nanoparticles comprise 59 mol %cationic lipid COATSOME® SS-EC (former name: SS-33/4PE-15 as apparentfrom the examples section; NOF Corporation, Tokyo, Japan), 29.3 mol %cholesterol as steroid, 10 mol % DPhyPE as neutral lipid/phospholipidand 1.7 mol % DMG-PEG 2000 as polymer conjugated lipid. A furtherinventive advantage connected with the use of DPhyPE is the highcapacity for fusogenicity due to its bulky tails, whereby it is able tofuse at a high level with endosomal lipids. For “GN01”, N/P (lipid tonucleic acid, e.g RNA mol ratio) preferably is 14 and total lipid/RNAmass ratio preferably is 40 (m/m).

In other embodiments, the at least one RNA, preferably the at least onemRNA is complexed with one or more lipids thereby forming lipidnanoparticles (LNP), wherein the LNP comprises

I at least one cationic lipid;

Ii at least one neutral lipid;

Iii at least one steroid or steroid analogue; and

Iiii at least one PEG-lipid as defined herein,

wherein the cationic lipid is DLin-KC2-DMA (50 mol %) or DLin-MC3-DMA(50 mol %), the neutral lipid is DSPC (10 mol %), the PEG lipid isPEG-DOMG (1.5 mol %) and the structural lipid is cholesterol (38.5 mol%).

In other embodiments, the at least one RNA, preferably the at least onemRNA is complexed with one or more lipids thereby forming lipidnanoparticles (LNP), wherein the LNP comprises SS15/Chol/DOPE (orDOPC)/DSG-5000 at mol % 50/38.5/10/1.5.

In other embodiments, the RNA of the invention may be formulated inliposomes, e.g. in liposomes as described in WO2019/222424,WO2019/226925, WO2019/232095, WO2019/232097, or WO2019/232208, thedisclosure of WO2019/222424, WO2019/226925, WO2019/232095,WO2019/232097, or WO2019/232208 relating to liposomes or lipid-basedcarrier molecules herewith incorporated by reference.

In various embodiments, LNPs that suitably encapsulates the at least oneRNA of the invention have a mean diameter of from about 50 nm to about200 nm, from about 60 nm to about 200 nm, from about 70 nm to about 200nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm,from about 90 nm to about 190 nm, from about 90 nm to about 180 nm, fromabout 90 nm to about 170 nm, from about 90 nm to about 160 nm, fromabout 90 nm to about 150 nm, from about 90 nm to about 140 nm, fromabout 90 nm to about 130 nm, from about 90 nm to about 120 nm, fromabout 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or200 nm and are substantially non-toxic. As used herein, the meandiameter may be represented by the z-average as determined by dynamiclight scattering as commonly known in the art.

The polydispersity index (PDI) of the nanoparticles is typically in therange of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2.Typically, the PDI is determined by dynamic light scattering.

In another preferred embodiment of the invention the lipid nanoparticleshave a hydrodynamic diameter in the range from about 50 nm to about 300nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150nm, or from about 60 nm to about 120 nm, respectively.

In another preferred embodiment of the invention the lipid nanoparticleshave a hydrodynamic diameter in the range from about 50 nm to about 300nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150nm, or from about 60 nm to about 120 nm, respectively.

In embodiments where more than one ora plurality, e.g. 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 of RNA species of the invention arecomprised in the composition, said more than one or said plurality e.g.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of RNA species of theinvention may be complexed within one or more lipids thereby formingLNPs comprising more than one or a plurality, e.g. 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 of different RNA species.

According to a preferred embodiment the LNPs preferably encapsulating orcomprising RNA are purified by at least one purification step,preferably by at least one step of TFF and/or at least one step ofclarification and/or at least one step of filtration. This purificationparticularly leads to reducing the amount of ethanol in the composition,which has been used for the lipid formulation.

In this context it is particularly preferred that the compositioncomprises after purification less than about 500 ppM ethanol, preferablyless than about 50 ppM ethanol, more preferably less than about 5 ppMethanol.

In embodiments, the LNPs described herein may be lyophilized in order toimprove storage stability of the formulation and/or the RNA. Inembodiments, the LNPs described herein may be spray dried in order toimprove storage stability of the formulation and/or the nucleic acid.Lyoprotectants for lyophilization and or spray drying may be selectedfrom trehalose, sucrose, mannose, dextran and inulin. A preferredlyoprotectant is sucrose, optionally comprising a further lyoprotectant.A further preferred lyoprotectant is trehalose, optionally comprising afurther lyoprotectant.

Accordingly, the composition, e.g. the composition comprising LNPs islyophilized (e.g. according to WO2016/165831 or WO2011/069586, each ofwhich is hereby incorporated in its entirety by reference) to yield atemperature stable dried nucleic acid (powder) composition as definedherein (e.g. RNA or DNA). The composition, e.g. the compositioncomprising LNPs may also be dried using spray-drying or spray-freezedrying (e.g. according to WO2016/184575 or WO2016/184576) to yield atemperature stable composition (powder) as defined herein.

Accordingly, in preferred embodiments, the composition is a driedcomposition.

The term “dried composition” as used herein has to be understood ascomposition that has been lyophilized, or spray-dried, or spray-freezedried as defined above to obtain a temperature stable dried composition(powder) e.g. comprising LNP complexed RNA (as defined above).

According to further embodiments, the composition of the second aspectmay comprise at least one adjuvant.

Suitably, the adjuvant is preferably added to enhance theimmunostimulatory properties of the composition.

The term “adjuvant” as used herein will be recognized and understood bythe person of ordinary skill in the art, and is for example intended torefer to a pharmacological and/or immunological agent that may modify,e.g. enhance, the effect of other agents or that may be suitable tosupport administration and delivery of the composition. The term“adjuvant” refers to a broad spectrum of substances. Typically, thesesubstances are able to increase the immunogenicity of antigens. Forexample, adjuvants may be recognized by the innate immune systems and,e.g., may elicit an innate immune response (that is, a non-specificimmune response). “Adjuvants” typically do not elicit an adaptive immuneresponse. In the context of the invention, adjuvants may enhance theeffect of the antigenic peptide or protein provided by the nucleic acid.In that context, the at least one adjuvant may be selected from anyadjuvant known to a skilled person and suitable for the present case,i.e. supporting the induction of an immune response in a subject, e.g.in a human subject.

Accordingly, the composition of the second aspect may comprise at leastone adjuvant, wherein the at least one adjuvant may be suitably selectedfrom any adjuvant provided in WO2016/203025, which is herebyincorporated by reference. Adjuvants disclosed in any of the claims 2 to17 of WO2016/203025, preferably adjuvants disclosed in claim 17 ofWO2016/203025 are particularly suitable, the specific content relatingthereto herewith incorporated by reference. Adjuvants may suitably usedand comprised in the composition of the second aspect, or the vaccine ofthe forth aspect, to e.g. reduce the amount of nucleic acid required fora sufficient immune response against the encoded protein and/or toimprove the efficacy of the composition/the vaccine fortreatment/vaccination of the elderly. A suitable adjuvant in the contextof a coronavirus composition or vaccine (in particular for compositionscomprising a polypeptide of the third aspect) may be a Toll-likereceptor 9 (TLR9) agonist adjuvant, CpG 1018™.

The composition of the second aspect may comprise, besides thecomponents specified herein, at least one further component which may beselected from the group consisting of further antigens (e.g. in the formof a peptide or protein, preferably derived from a coronavirus) orfurther antigen-encoding nucleic acids (preferably encoding peptide orprotein, preferably derived from a coronavirus); a furtherimmunotherapeutic agent; one or more auxiliary substances (cytokines,such as monokines, lymphokines, interleukins or chemokines); or anyfurther compound, which is known to be immune stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors; and/or anadjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA), e.g.CpG-RNA etc.

In preferred embodiments, the composition comprising lipid-basedcarriers (e.g. LNPs) encapsulating the at least one RNA is stable afterstorage as a liquid, for example stable for at least 2 weeks afterstorage as a liquid at temperatures of about 5° C.

As used herein, “stable” refers to a liquid composition comprisinglipid-based carriers (e.g. LNPs) encapsulating an RNA where the measuredvalues for various physiochemical parameters are within a defined rangeafter storage. In one embodiment, the liquid composition comprisinglipid-based carriers encapsulating an RNA is analyzed to assessstability according to various parameters. Suitable stability parametersinclude, without limitation, RNA integrity, Z-average particle size,polydispersity index (PDI), the amount of free RNA in the liquidcomposition, encapsulation efficiency of the RNA (proportion of the RNAin percent incorporated with lipid-based carriers), shape and morphologyof the lipid-based carriers encapsulating an RNA, pH, osmolality, orturbidity. Further, “stable” refers to a liquid composition comprisinglipid-based carriers encapsulating an RNA where the measured values forvarious functional parameters are within a defined range after storage.In one embodiment, the liquid composition comprising lipid-basedcarriers encapsulating an RNA is analyzed to assess the potency of theliquid composition including for example the expression of the encodedpeptide or protein, the induction of specific antibody titers, theinduction of neutralizing antibody titers, the induction of T-cell, thereactogenicity of the liquid composition including for example theinduction of innate immune responses ect.

In preferred embodiments, the term “stable” refers to RNA integrity.

The term “RNA integrity” generally describes whether the complete RNAsequence is present in the liquid composition. Low RNA integrity couldbe due to, amongst others, RNA degradation, RNA cleavage, incorrect orincomplete chemical synthesis of the RNA, incorrect base pairing,integration of modified nucleotides or the modification of alreadyintegrated nucleotides, lack of capping or incomplete capping, lack ofpolyadenylation or incomplete polyadenylation, or incomplete RNA invitro transcription. RNA is a fragile molecule that can easily degrade,which may be caused e.g. by temperature, ribonucleases, pH or otherfactors (e.g. nucleophilic attacks, hydrolysis etc.), which may reducethe RNA integrity and, consequently, the functionality of the RNA.

In preferred embodiments, the RNA of a composition has an RNA integrityof at least about 50%, preferably of at least about 60%, more preferablyof at least about 70%, most preferably of at least about 80% or about90%. RNA is suitably determined using analytical HPLC, preferablyanalytical RP-HPLC.

The skilled person can choose from a variety of differentchromatographic or electrophoretic methods for determining an RNAintegrity. Chromatographic and electrophoretic methods are well-known inthe art. In case chromatography is used (e.g. RP-HPLC), the analysis ofthe integrity of the RNA may be based on determining the peak area (or“area under the peak”) of the full length RNA in a correspondingchromatogram. The peak area may be determined by any suitable softwarewhich evaluates the signals of the detector system. The process ofdetermining the peak area is also referred to as integration. The peakarea representing the full length RNA is typically set in relation tothe peak area of the total RNA in a respective sample. The RNA integritymay be expressed in % RNA integrity.

In the context of aspects of the invention, RNA integrity may bedetermined using analytical (RP)HPLC. Typically, a test sample of theliquid composition comprising lipid based carrier encapsulating RNA maybe treated with a detergent (e.g. about 2% Triton X100) to dissociatethe lipid based carrier and to release the encapsulated RNA. Thereleased RNA may be captured using suitable binding compounds, e.g.Agencourt AMPure XP beads (Beckman Coulter, Brea, Calif., USA)essentially according to the manufacturer's instructions. Followingpreparation of the RNA sample, analytical (RP)HPLC may be performed todetermine the integrity of RNA. Typically, for determining RNAintegrity, the RNA samples may be diluted to a concentration of 0.1 g/lusing e.g. water for injection (WFI). About 10 μl of the diluted RNAsample may be injected into an HPLC column (e.g. a monolithicpoly(styrene-divinylbenzene) matrix). Analytical (RP)HPLC may beperformed using standard conditions, for example: Gradient 1: Buffer A(0.1M TEAA (pH 7.0)); Buffer B (0.1M TEAA (pH 7.0) containing 25%acetonitrile). Starting at 30% buffer B the gradient extended to 32%buffer B in 2 min, followed by an extension to 55% buffer B over 15minutes at a flow rate of lml/min. HPLC chromatograms are typicallyrecorded at a wavelength of 260 nm. The obtained chromatograms may beevaluated using a software and the relative peak area may be determinedin percent (%) as commonly known in the art. The relative peak areaindicates the amount of RNA that has 100% RNA integrity. Since theamount of the RNA injected into the HPLC is typically known, theanalysis of the relative peak area provides information on the integrityof the RNA. Thus, if e.g. 100 ng RNA have been injected in total, and100 ng are determined as the relative peak area, the RNA integrity wouldbe 100%. If, for example, the relative peak area would correspond to 80ng, the RNA integrity would be 80%. Accordingly, RNA integrity in thecontext of the invention is determined using analytical HPLC, preferablyanalytical RP-HPLC.

In preferred embodiments, 80% of RNA comprised in the liquid compositionis encapsulated, preferably 85% of the RNA comprised in the compositionis encapsulated, 90% of the RNA comprised in the composition isencapsulated, most preferably95% or more of the RNA comprised in thecomposition is encapsulated. The percentage of encapsulation may bedetermined by a Ribogreen assay as known in the art.

In embodiments, the composition comprises at least one antagonist of atleast one RNA sensing pattern recognition receptor. Such an antagonistmay preferably be co-formulated in lipid-based carriers as definedherein.

Suitable antagonist of at least one RNA sensing pattern recognitionreceptor are disclosed in PCT patent application PCT/EP2020/072516, thefull disclosure herewith incorporated by reference. In particular, thedisclosure relating to suitable antagonist of at least one RNA sensingpattern recognition receptors as defined in any one of the claims 1 to94 of PCT/EP2020/072516 are incorporated.

In preferred embodiments, the composition comprises at least oneantagonist of at least one RNA sensing pattern recognition receptorselected from a Toll-like receptor, preferably TLR7 and/or TLR8.

In embodiments, the at least one antagonist of at least one RNA sensingpattern recognition receptor is selected from a nucleotide, a nucleotideanalog, a nucleic acid, a peptide, a protein, a small molecule, a lipid,or a fragment, variant or derivative of any of these.

In preferred embodiments, the at least one antagonist of at least oneRNA sensing pattern recognition receptor is a single strandedoligonucleotide, preferably a single stranded RNA Oligonucleotide.

In embodiments, the antagonist of at least one RNA sensing patternrecognition receptor is a single stranded oligonucleotide that comprisesor consists of a nucleic acid sequence identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 85-212 of PCT/EP2020/072516, or fragments ofany of these sequences.

In preferred embodiments, the antagonist of at least one RNA sensingpattern recognition receptor is a single stranded oligonucleotide thatcomprises or consists of a nucleic acid sequence identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 85-87, 149-212 of PCT/EP2020/072516, orfragments of any of these sequences.

A particularly preferred antagonist of at least one RNA sensing patternrecognition receptor in the context of the invention is 5′-GAG CGmGCCA-3′ (SEQ ID NO: 85 of PCT/EP2020/072516), ora fragment thereof.

In embodiments, the molar ratio of the at least one antagonist of atleast one RNA sensing pattern recognition receptor as defined herein tothe at least one nucleic acid, preferably RNA encoding a SRAS-CoV-2antigenic peptide or protein as defined herein suitably ranges fromabout 1:1, to about 100:1, or ranges from about 20:1, to about 80:1.

In embodiments, the wherein the weight to weight ratio of the at leastone antagonist of at least one RNA sensing pattern recognition receptoras defined herein to the at least one nucleic acid, preferably RNAencoding a SRAS-CoV-2 antigenic peptide or protein as defined hereinsuitably ranges from about 1:1, to about 1:30, or ranges from about 1:2,to about 1:10.

Vaccine:

In a third aspect, the present invention provides a vaccine, for examplea vaccine against a SARS-CoV-2 (formerly nCoV-2019) coronavirus causingCOVID-19 disease. The vaccine may be effective against multipleSARS-CoV-2 coronoaviruses. The vaccine may also be effective againstboth one or more SARS-CoV-2 coronaviruses and one or morenon-coronaviruses (e.g., the vaccine may be effective against both aSARS-CoV-2 virus and an influenza virus)>

In preferred embodiments of the fourth aspect, the vaccine comprises atleast one nucleic acid (e.g. DNA or RNA), preferably at least one RNA ofthe first aspect, or the composition of the second aspect.

In other embodiments, the vaccine comprises at least one polypeptide asdefined in the third aspect.

In other embodiments, the vaccine comprises at least one plasmid DNA oradenovirus DNA as defined in the first aspect.

Notably, embodiments relating to the composition of the second aspectmay likewise be read on and be understood as suitable embodiments of thevaccine of the fourth aspect. Also, embodiments relating to the vaccineof the fourth aspect may likewise be read on and be understood assuitable embodiments of the composition of the second aspect.Furthermore, features and embodiments described in the context of thefirst aspect (the nucleic acid of the invention) have to be read on andhave to be understood as suitable embodiments of the composition of thefourth aspect.

The term “vaccine” will be recognized and understood by the person ofordinary skill in the art, and is for example intended to be aprophylactic or therapeutic material providing at least one epitope orantigen, preferably an immunogen. In the context of the invention theantigen or antigenic function is suitably provided by the inventivenucleic acid of the first aspect (said nucleic acid comprising a codingsequence encoding a antigenic peptide or protein derived from aSARS-CoV-2 coronavirus) or the composition of the second aspect(comprising at least one nucleic acid of the first aspect). In otherembodiments, the antigen or antigenic function is provided by theinventive polypeptide of the third aspect.

In preferred embodiments, the vaccine, or the composition of the secondaspect, elicits an adaptive immune response, preferably an adaptiveimmune response against a coronavirus, preferably against SARS-CoV-2coronavirus.

In particularly preferred embodiments, the vaccine, or the compositionof the second aspect, elicits functional antibodies that can effectivelyneutralize the virus, preferably SARS-CoV-2 coronavirus.

In further preferred embodiments, the vaccine, or the composition of thesecond aspect, elicits mucosal IgA immunity by inducing of mucosal IgAantibodies.

In particularly preferred embodiments, the vaccine, or the compositionof the second aspect, elicits functional antibodies that can effectivelyneutralize the virus, preferably SARS-CoV-2 coronavirus.

In further particularly preferred embodiments, the vaccine, or thecomposition of the second aspect, induces broad, functional cellularT-cell responses against coronavirus, preferably against SARS-CoV-2coronavirus.

In further particularly preferred embodiments, the vaccine, or thecomposition of the second aspect, induces a well-balanced B cell and Tcell response against coronavirus, preferably against SARS-CoV-2coronavirus.

According to a preferred embodiment, the vaccine as defined herein mayfurther comprise a pharmaceutically acceptable carrier and optionally atleast one adjuvant as specified in the context of the second aspect.

Suitable adjuvants in that context may be selected from adjuvantsdisclosed in claim 17 of WO2016/203025.

In a preferred embodiment, the vaccine is a monovalent vaccine.

The terms “monovalent vaccine”, “monovalent composition” “univalentvaccine” or “univalent composition” will be recognized and understood bythe person of ordinary skill in the art, and are e.g. intended to referto a composition or a vaccine comprising only one antigen or antigenconstruct from a pathogen. Accordingly, said vaccine or compositioncomprises only one nucleic acid species encoding a single antigen orantigen construct of a single organism. The term “monovalent vaccine”includes the immunization against a single valence. In the context ofthe invention, a monovalent SARS-CoV-2 coronavirus vaccine orcomposition would comprise at least one nucleic acid encoding one singleantigenic peptide or protein derived from one specific SARS-CoV-2coronavirus.

In embodiments, the vaccine is a polyvalent vaccine comprising aplurality or at least more than one of the nucleic acid species definedin the context of the first aspect. Embodiments relating to a polyvalentcomposition as disclosed in the context of the second aspect maylikewise be read on and be understood as suitable embodiments of thepolyvalent vaccine.

The terms “polyvalent vaccine”, “polyvalent composition” “multivalentvaccine” or “multivalent composition” will be recognized and understoodby the person of ordinary skill in the art, and are e.g. intended torefer to a composition or a vaccine comprising antigens from more thanone virus (e.g. different SARS-CoV-2 coronavirus isolates), orcomprising different antigens or antigen constructs of the sameSARS-CoV-2 coronavirus, or any combination thereof. The terms describethat said vaccine or composition has more than one valence. In thecontext of the invention, a polyvalent SARS-CoV-2 coronavirus vaccinewould comprise nucleic acid sequences encoding antigenic peptides orproteins derived from several different SARS-CoV-2 coronavirus (e.g.different SARS-CoV-2 coronavirus isolates) or comprising nucleic acidsequences encoding different antigens or antigen constructs from thesame SARS-CoV-2 coronavirus, or a combination thereof.

In preferred embodiments, the polyvalent or multivalent vaccinecomprises at least one polyvalent composition as defined in the secondaspect. Particularly preferred are polyvalent compositions as defined insection “Multivalent compositions of the invention”.

In some embodiments, the vaccine comprises at least one antagonist of atleast one RNA sensing pattern recognition receptor as defined in thesecond aspect.

The vaccine typically comprises a safe and effective amount of nucleicacid (e.g. DNA or RNA), preferably RNA of the first aspect orcomposition of the second aspect (or the polypeptide of the thirdaspect). As used herein, “safe and effective amount” means an amount ofnucleic acid or composition sufficient to significantly induce apositive modification of a disease or disorder related to an infectionwith coronavirus, preferably SARS-CoV-2 coronavirus. At the same time, a“safe and effective amount” is small enough to avoid seriousside-effects. In relation to the nucleic acid, composition, or vaccineof the present invention, the expression “safe and effective amount”preferably means an amount of nucleic acid, composition, or vaccine thatis suitable for stimulating the adaptive immune system againstcoronavirus in such a manner that no excessive or damaging immunereactions (e.g. innate immune responses) are achieved.

A “safe and effective amount” of the nucleic acid, composition, orvaccine as defined above will vary in connection with the particularcondition to be treated and also with the age and physical condition ofthe patient to be treated, the severity of the condition, the durationof the treatment, the nature of the accompanying therapy, of theparticular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the skilled person.Moreover, the “safe and effective amount” of the nucleic acid, thecomposition, or vaccine may depend from application/delivery route(intradermal, intramuscular, intranasal), application device (jetinjection, needle injection, microneedle patch, electroporation device)and/or complexation/formulation (protamine complexation or LNPencapsulation, DNA or RNA). Moreover, the “safe and effective amount” ofthe nucleic acid, the composition, or the vaccine may depend from thephysical condition of the treated subject (infant, pregnant women,immunocompromised human subject etc.).

The vaccine can be used according to the invention for human medicalpurposes and also for veterinary medical purposes (mammals, vertebrates,or avian species).

The pharmaceutically acceptable carrier as used herein preferablyincludes the liquid or non-liquid basis of the vaccine. If the vaccineis provided in liquid form, the carrier will be water, typicallypyrogen-free water; isotonic saline or buffered (aqueous) solutions,e.g. phosphate, citrate etc. buffered solutions. Preferably,Ringer-Lactate solution is used as a liquid basis for the vaccine or thecomposition according to the invention as described in WO2006/122828,the disclosure relating to suitable buffered solutions incorporatedherewith by reference. Other preferred solutions used as a liquid basisfor the vaccine or the composition, in particular forcompositions/vaccines comprising LNPs, comprise sucrose and/ortrehalose.

The choice of a pharmaceutically acceptable carrier as defined herein isdetermined, in principle, by the manner, in which the pharmaceuticalcomposition(s) or vaccine according to the invention is administered.The vaccine is preferably administered locally. Routes for localadministration in general include, for example, topical administrationroutes but also intradermal, transdermal, subcutaneous, or intramuscularinjections or intralesional, intracranial, intrapulmonal, intracardial,intraarticular and sublingual injections. More preferably, compositionor vaccines according to the present invention may be administered by anintradermal, subcutaneous, or intramuscular route, preferably byinjection, which may be needle-free and/or needle injection. Preferredin the context of the invention is intramuscular injection.Compositions/vaccines are therefore preferably formulated in liquid orsolid form. The suitable amount of the vaccine or composition accordingto the invention to be administered can be determined by routineexperiments, e.g. by using animal models. Such models include, withoutimplying any limitation, rabbit, sheep, mouse, rat, dog and non-humanprimate models. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4.

The vaccine or composition as defined herein may comprise one or moreauxiliary substances or adjuvants as defined above in order to furtherincrease the immunogenicity. A synergistic action of the nucleic acidcontained in the composition/vaccine and of an auxiliary substance,which may be optionally be co-formulated (or separately formulated) withthe vaccine or composition as described above, is preferably achievedthereby. Such immunogenicity increasing agents or compounds may beprovided separately (not co-formulated with the vaccine or composition)and administered individually.

The vaccine is preferably provided in lyophilized or spray-dried form(as described in the context of the second aspect). Such a lyophilizedor spray-dried vaccine typically comprises trehalose and/or sucrose andis re-constituted in a suitable liquid buffer before administration to asubject. In some aspects, a lyophilized vaccine of the embodimentscomprises mRNA of the embodiments complexed with LNPs. In some aspects,a lyophilized composition has a water content of less than about 10%.For example, a lyophilized composition can have a water content of about0.1% to 10%, 0.1% to 7.5%, or 0.5% to 7.5%, preferably the lyophilizedcomposition has a water content of about 0.5% to about 5.0%.

In preferred embodiments administration of a therapeutically effectiveamount of the nucleic acid, the composition, the polypeptide, thevaccine to a subject induces a neutralizing antibody titer againtsSARS-CoV-2 coronavirus in the subject.

In some embodiments, the neutralizing antibody titer is at least 100neutralizing units per milliliter (NU/mL), at least 500 NU/mL, or atleast 1000 NU/mL.

In some embodiments, detectable levels of the coronavirus antigen areproduced in the subject at about 1 to about 72 hours post administrationof the nucleic acid, the composition, the polypeptide, or the vaccine.

In some embodiments, a neutralizing antibody titer (against coronavirus)of at least 100 NU/ml, at least 500 NU/ml, or at least 1000 NU/ml isproduced in the serum of the subject at about 1 day to about 72 dayspost administration of the nucleic acid, the composition, thepolypeptide, or the vaccine.

In some embodiments, the neutralizing antibody titer is sufficient toreduce coronavirus infection by at least 50% relative to a neutralizingantibody titer of an unvaccinated control subject or relative to aneutralizing antibody titer of a subject vaccinated with a liveattenuated viral vaccine, an inactivated viral vaccine, or a proteinsubunit viral vaccine.

In some embodiments, the neutralizing antibody titer and/or a T cellimmune response is sufficient to reduce the rate of asymptomatic viralinfection relative to the neutralizing antibody titer of unvaccinatedcontrol subjects.

In some embodiments, the neutralizing antibody titer and/or a T cellimmune response is sufficient to prevent viral latency in the subject.

In some embodiments, the neutralizing antibody titer is sufficient toblock fusion of virus with epithelial cells of the subject.

In some embodiments, the neutralizing antibody titer is induced within20 days following a single 1 ug-100 ug dose of the nucleic acid, thecomposition, the polypeptide, or the vaccine, or within 40 daysfollowing a second 1 ug-100 μg dose of the nucleic acid, thecomposition, the polypeptide, or the vaccine.

In preferred embodiments, administration of a therapeutically effectiveamount of the nucleic acid, the composition, the polypeptide, or thevaccine to a subject induces a T cell immune response againstcoronavirus in the subject. In preferred embodiments, the T cell immuneresponse comprises a CD4+ T cell immune response and/or a CD8+ T cellimmune response.

Kit or Kit of Parts, Application, Medical Uses, Method of Treatment:

In a fourth aspect, the present invention provides a kit or kit of partssuitable for treating or preventing a coronavirus infection. Preferably,said kit or kit of parts is suitable for treating or preventing acoronavirus, preferably a SARS-CoV-2 (formerly nCoV-2019) coronavirusinfection.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect may likewise be read on andbe understood as suitable embodiments of the kit or kit of parts of thefifth aspect of the invention.

In preferred embodiments, the kit or kit of parts comprises at least onenucleic acid (e.g. RNA or DNA), preferably at least one RNA of the firstaspect, at least one composition of the second aspect, and/or at leastone polypeptide of the third aspect, and/or at least one vaccine of thefourth aspect.

In embodiments, the kit or kit of parts comprises at least one DNA asdefined in the first aspect, e.g. at least one plasmid DNA and/or atleast one adenovirus DNA.

In embodiments, the kit or kit of parts comprises at least onepolypeptide as defined in the third aspect.

In addition, the kit or kit of parts may comprise a liquid vehicle forsolubilising, and/or technical instructions providing information onadministration and dosage of the components.

The kit may further comprise additional components as described in thecontext of the composition of the second aspect, and/or the vaccine ofthe fourth aspect.

The technical instructions of said kit may contain information aboutadministration and dosage and patient groups. Such kits, preferably kitsof parts, may be applied e.g. for any of the applications or usesmentioned herein, preferably for the use of the nucleic acid of thefirst aspect, the composition of the second aspect, the polypeptide ofthe third aspect, or the vaccine of the fourth aspect, for the treatmentor prophylaxis of an infection or diseases caused by a coronavirus,preferably SARS-CoV-2 coronavirus, or disorders related thereto.

Preferably, the nucleic acid, the composition, the polypeptide, or thevaccine is provided in a separate part of the kit, wherein the nucleicacid, the composition, the polypeptide, or the vaccine is preferablylyophilised.

The kit may further contain as a part a vehicle (e.g. buffer solution)for solubilising the nucleic acid, the composition, the polypeptide, orthe vaccine.

In preferred embodiments, the kit or kit of parts as defined hereincomprises Ringer lactate solution.

In preferred embodiments, the kit or kit of parts as defined hereincomprises a multidose container for administration of thecomposition/the vaccine.

Any of the above kits may be used in a treatment or prophylaxis asdefined herein. More preferably, any of the above kits may be used as avaccine, preferably a vaccine against infections caused by acoronavirus, preferably caused by SARS-CoV-2 coronavirus.

In preferred embodiments, the kit or kit of parts comprises thefollowing components:

-   -   a) at least one container or vial comprising a composition or        SARS-CoV-2 vaccine as defined herein, wherein the composition or        SARS-CoV-2 vaccine has a nucleic acid concentration, preferably        an RNA concentration in a range of about 100 μg/ml to about 1        mg/ml, preferably in a range of about 100 μg/ml to about 500        μg/ml, e.g. about 270 μg/ml.    -   b) at least one dilution container or vial comprising a sterile        dilution buffer, suitably a buffer comprising NaCl, optionally        comprising a preservative;    -   c) at least one means for transferring the composition or        vaccine from the storage container to the dilution container;        and    -   d) at least one syringe for administering the final diluted        composition or vaccine to a subject, preferably configured for        intramuscular administration to a human subject, wherein the        final diluted composition or vaccine has a nucleic acid        concentration, preferably an RNA concentration in a range of        about 10 μg/ml to about 100 μg/ml, preferably in a range of        about 10 μg/ml to about 50 μg/ml, e.g. about 24 μg/ml

In an embodiment, the kit or kit of parts comprises more than onemRNA-based SARS-CoV-2 composition/vaccine, preferably

-   -   at least one vaccine as defined herein provided in a first vial        or container, wherein the vaccine comprises at least one nucleic        acid, preferably RNA, which is identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a nucleic acid sequence of SEQ ID NOs:        163, 149 or 24837, preferably formulated in lipid nanoparticles        (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid 111-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 22792, 22794, 22796, 22798,        22800, 22802, 22804, 22806, 22808, 22810, 22812, 23529-23534,        27386-27408, 23535-23552, 27409-27431, 23590-23606, 27478-27500,        28736-28776, 28638-28686, 28777-28825, 28925-28928, 28933-28936        preferably formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)). Preferably, the nucleic acid, preferably mRNA is        not chemically modified; and/or.    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 27394, 27417, 27486, 28762,        28650-28655, 28789-28794, preferably formulated in lipid        nanoparticles (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 27394, 27417, 27486, 28762,        preferably formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)). Preferably, the nucleic acid, preferably mRNA is        not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 28639-28642, 28778-28781,        28925-28928, 28933-28936 preferably formulated in lipid        nanoparticles (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified

In an embodiment, the kit or kit of parts comprises more than onemRNA-based SARS-CoV-2 composition/vaccine, preferably

-   -   at least one vaccine as defined herein provided in a first vial        or container, wherein the vaccine comprises at least one nucleic        acid, preferably RNA, which is identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a nucleic acid sequence of SEQ ID NOs:        163, 149 or 24837, preferably formulated in lipid nanoparticles        (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 24837-24854, 27524-27546,        24855-24872, 27547-27569, 24909-24926, 27616-27638, 28827-28866,        28687-28735, 28867-28915, 28929-28932, 28937-28940 preferably        formulated in lipid nanoparticles (LNPs), which have a molar        ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)). Preferably, the nucleic acid, preferably mRNA is        not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 27532, 27555, 27624, 28852,        28699-28704, 28879-28884, preferably formulated in lipid        nanoparticles (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 27532, 27555, 27624, 28852,        preferably formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)). Preferably, the nucleic acid, preferably mRNA is        not chemically modified; and/or    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NOs: 28688-28691, 28868-28871,        28929-28932, 28937-28940 preferably formulated in lipid        nanoparticles (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified.

In an embodiment, the kit or kit of parts comprises two differentSARS-CoV-2 vaccines for prime vaccination and boost vaccination:

-   -   at least one prime vaccine as defined herein provided in a first        vial or container, wherein the vaccine is an mRNA-based        SARS-CoV-2 vaccine as defined herein; and    -   at least one boost vaccine as defined herein provided in a first        vial or container, wherein the composition/vaccine is an        adenovirus-based SARS-CoV-2 vaccine as defined herein.

In an embodiment, the kit or kit of parts comprises two differentSARS-CoV-2 vaccines for prime vaccination and boost vaccination:

-   -   at least one boost vaccine as defined herein provided in a first        vial or container, wherein the vaccine is an mRNA-based        SARS-CoV-2 vaccine as defined herein; and    -   at least one prime vaccine as defined herein provided in a first        vial or container, wherein the composition/vaccine is an        adenovirus-based SARS-CoV-2 vaccine as defined herein.

Combination:

A fifth aspect relates to a combination of at least two nucleic acidsequences as defined in the first aspect, at least two compositions asdefined in the context of the second aspect, at least two polypeptidesas defined in the third aspect, at least two vaccines as defined in thecontext of the fourth aspect, or at least two kits as defined in thefifth aspect.

In the context of the present invention, the term “combination”preferably means a combined occurrence of at least two components,preferably at least two nucleic acid sequences as defined in the firstaspect, at least two compositions as defined in the context of thesecond aspect, at least two polypeptides as defined in the third aspect,at least two vaccines as defined in the context of the fourth aspect, orat least two kits as defined in the fifth aspect. The components of sucha combination may occur as separate entities. Thus, the administrationof the components of the combination may occur either simultaneously ortimely staggered, either at the same site of administration or atdifferent sites of administration.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, or the kit or kit of partsof the fifth aspect may likewise be read on and be understood assuitable embodiments of the components of the combination of the sixthaspect.

In embodiments, the combination may comprise a plurality or at leastmore than one of the nucleic acid species, e.g. RNA species as definedin the context of the first aspect of the invention, wherein the nucleicacid species are provided as separate components.

Preferably, the combination as defined herein may comprise 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different nucleic acids e.g. RNA species asdefined in the context of the first aspect of the invention; 2, 3, 4, 5,6, 7, 8, 9, or 10 different compositions as defined in the context ofthe second aspect of the invention; 2, 3, 4, 5, 6, 7, 8, 9, or 10different polypeptides as defined in the context of the third aspect ofthe invention; 2, 3, 4, 5, 6, 7, 8, 9, or 10 different vaccines asdefined in the context of the third aspect of the invention, wherein thenucleic acid species, compositions, polypeptides, vaccines are providedas separate components.

In embodiments, the combination comprises 2, 3, 4 or 5 RNAs comprised inseparate components, preferably RNA species, wherein said nucleic acidspecies comprise or consist of a nucleic acid sequence which isidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 149, 22792,22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810, 22812,23529-23534, 27386-27408, 23535-23552, 27409-27431, 23590-23606,27478-27500, 28736-28776, 28638-28686, 28777-28825, 28925-28928,28933-28936 and, optionally, at least one pharmaceutically acceptablecarrier or excipient, wherein each of the 2, 3, 4 or 5 nucleic acidspecies encode a different antigenic peptide or protein of a SARS-CoV-2coronavirus.

In embodiments, the combination comprises 2, 3, 4 or 5 RNAs comprised inseparate components, preferably RNA species, wherein said nucleic acidspecies comprise or consist of a nucleic acid sequence which isidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 24837-24854,27524-27546, 24855-24872, 27547-27569, 24909-24926, 27616-27638,28827-28866, 28687-28735, 28867-28915, 28929-28932, 28937-28940 and,optionally, at least one pharmaceutically acceptable carrier orexcipient, wherein each of the 2, 3, 4 or 5 nucleic acid species encodea different antigenic peptide or protein of a SARS-CoV-2 coronavirus.

In the following, particularly preferred embodiments of a combinationare provided, wherein each component of the combination is provided asseparate entities.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, compositions, vaccines of thecombination each encode a different prefusion stabilized spike protein(as defined in the first aspect). Preferably, stabilization of theprefusion conformation is obtained by introducing two consecutiveproline substitutions at residues K986 and V987 in the spike protein(Amino acid positions according to reference SEQ ID NO: 1). Accordingly,in preferred embodiments, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10pre-fusion stabilized spike proteins (S_stab) each comprises at leastone pre-fusion stabilizing mutation, wherein the at least one pre-fusionstabilizing mutation comprises the following amino acid substitutions:K986P and V987P (amino acid positions according to reference SEQ ID NO:1).

Accordingly, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, compositions, vaccines of thecombination each encode a different prefusion stabilized spike protein,wherein the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more stabilizedspike proteins are selected from amino acid sequences being identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10,22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756,22758, 22959-22964, 27087-27109, 28540-28588, 28917-28920 or animmunogenic fragment or immunogenic variant of any of these.

In preferred embodiments, the combination comprises one nucleic acidspecies, composition, vaccine comprising a coding sequence encoding anamino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   -   i) one nucleic acid species comprises a coding sequence encoding        an amino acid sequence being identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to any one of SEQ ID NOs: 22961; and/or    -   ii) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 22960;        and/or    -   iii) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 22963;        and/or    -   iv) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 22959;    -   v) one nucleic acid species comprises a coding sequence encoding        an amino acid sequence being identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to any one of SEQ ID NOs: 27070, 27093;    -   vi) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 27071,        27094;    -   vii) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 27072,        27095;    -   viii) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 27073,        27096, 28545; and/or    -   ix) one nucleic acid species comprises a coding sequence        encoding an amino acid sequence being identical or at least 70%,        80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% identical to any one of SEQ ID NOs: 22964.    -   x) one nucleic acid species comprises a coding sequence encoding        an amino acid sequence being identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to any one of SEQ ID NOs: 28541-28544,        28917-28920.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, composition, vaccine of the combinationcomprise nucleic acid coding sequences each encoding a differentprefusion stabilized spike protein, wherein the at least 2, 3, 4, 5, 6,7, 8, 9, 10 or even more nucleic acid coding sequences are selected fromnucleic acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 116, 136, 137, 146, 148, 149, 151,162, 163, 165, 22765, 22767, 22769, 22771, 22773, 22775, 22777, 22779,22781, 22783, 22785, 22792, 22794, 22796, 22798, 22800, 22802, 22804,22806, 22808, 22810, 22812, 22819, 22821, 22823, 22825, 22827, 22829,22831, 22833, 22835, 22837, 22839, 28916, 23089-23148, 23150-23184,23309-23368, 23370-23404, 23529-23588, 23590-23624, 24837-24944,27110-27907, 28589-28915, 28916, 28921-28940 or fragments or variants ofany of these. Preferably, each of the mRNA species comprise a cap1structure, and, optionally, each of the mRNA species do not comprisemodified nucleotides.

In a specific embodiment, a first component of the combination comprisesa viral vector vaccine/composition, such as an adenovirus vector basedvaccine, e.g., ADZ1222 or Ad26.COV-2.S, and a second component comprisesa nucleic acid based vaccine/composition, preferably an mRNA-basedvaccine as defined herein.

First and Second/Further Medical Use:

A further aspect relates to the first medical use of the providednucleic acid, composition, vaccine, kit, or combination.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, or the kit or kit of partsof the fifth aspect, or the combination may likewise be read on and beunderstood as suitable embodiments of medical uses of the invention.

Accordingly, the invention provides at least one nucleic acid (e.g. DNAor RNA), preferably RNA as defined in the first aspect for use as amedicament, the composition as defined in the second aspect for use as amedicament, the polypeptide as defined in the third aspect for use as amedicament, the vaccine as defined in the fourth aspect for use as amedicament, and the kit or kit of parts as defined in the fifth aspectfor use as a medicament, and the combination.

The present invention furthermore provides several applications and usesof the nucleic acid, composition, polypeptide, vaccine, or kit, orcombination.

In particular, nucleic acid (preferably RNA), composition, polypeptide,vaccine, or kit, or combination may be used for human medical purposesand also for veterinary medical purposes, preferably for human medicalpurposes.

In particular, nucleic acid (preferably RNA), composition, polypeptide,vaccine, or kit or kit of parts or combination is for use as amedicament for human medical purposes, wherein said nucleic acid(preferably RNA), composition, polypeptide, vaccine, or kit or kit ofparts may be suitable for young infants, newborns, immunocompromisedrecipients, as well as pregnant and breast-feeding women and elderlypeople. In particular, nucleic acid (preferably RNA), composition,polypeptide, vaccine, or kit or kit of parts is for use as a medicamentfor human medical purposes, wherein said nucleic acid (preferably RNA),composition, polypeptide, vaccine, or kit or kit of parts isparticularly suitable for elderly human subjects.

Said nucleic acid (preferably RNA), composition, polypeptide, vaccine,or kit or combination is for use as a medicament for human medicalpurposes, wherein said RNA, composition, vaccine, or the kit or kit ofparts may be particularly suitable for intramuscular injection orintradermal injection.

In yet another aspect, the invention relates to the second medical useof the provided nucleic acid, composition, polypeptide, vaccine, or kitor combination.

Accordingly, the invention provides at least one nucleic acid,preferably RNA as defined in the first aspect for treatment orprophylaxis of an infection with a coronavirus, preferably SARS-CoV-2coronavirus, or a disorder or a disease related to such an infection,such as COVID-19; a composition as defined in the second aspect fortreatment or prophylaxis of an infection with a coronavirus, preferablySARS-CoV-2 coronavirus, or a disorder or a disease related to such aninfection, such as COVID-19; a polypeptide as defined in the thirdaspect for treatment or prophylaxis of an infection with a coronavirus,preferably SARS-CoV-2 coronavirus, or a disorder or a disease related tosuch an infection, such as COVID-19; a vaccine as defined in the fourthaspect for treatment or prophylaxis of an infection with a coronavirus,preferably SARS-CoV-2 coronavirus, or a disorder or a disease related tosuch an infection, such as COVID-19; a kit or kit of parts as defined inthe fifth aspect for treatment or prophylaxis of an infection with acoronavirus, preferably SARS-CoV-2 coronavirus, or a disorder or adisease related to such an infection, such as COVID-19; a combination asdefined in the sixth aspect for treatment or prophylaxis of an infectionwith a coronavirus, preferably SARS-CoV-2 coronavirus, or a disorder ora disease related to such an infection, such as COVID-19.

In embodiments, the nucleic acid, preferably RNA of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, the vaccine of the fourth aspect, or the kit or kit of parts ofthe fifth aspect, or the combination of the sixth aspect, is for use inthe treatment or prophylaxis of an infection with a coronavirus,preferably with SARS-CoV-2 coronavirus.

Particularly, the nucleic acid, preferably RNA of the first aspect, thecomposition of the second aspect, the polypeptide of the third aspect,the vaccine of the fourth aspect, or the kit or kit of parts of thefifth aspect, or the combination of the sixth aspect, may be used in amethod of prophylactic (pre-exposure prophylaxis or post-exposureprophylaxis) and/or therapeutic treatment of infections caused by acoronavirus, preferably SARS-CoV-2 coronavirus.

Particularly, the nucleic acid, preferably RNA of the first aspect, thecomposition of the second aspect, the polypeptide of the third aspect,the vaccine of the fourth aspect, or the kit or kit of parts of thefifth aspect, or the combination of the sixth aspect, may be used in amethod of prophylactic (pre-exposure prophylaxis or post-exposureprophylaxis) and/or therapeutic treatment of COVID-19 disease caused bya SARS-CoV-2 coronavirus infection.

The nucleic acid, the composition, the polypeptide, or the vaccine, orthe combination may preferably be administered locally. In particular,composition or polypeptides or vaccines or combinations may beadministered by an intradermal, subcutaneous, intranasal, orintramuscular route. In embodiments, the inventive nucleic acid,composition, polypeptide, vaccine may be administered by conventionalneedle injection or needle-free jet injection. Preferred in that contextis intramuscular injection.

In embodiments where plasmid DNA is used and comprised in thecomposition or vaccine or combination, thecomposition/vaccine/combination may be administered by electroporationusing an electroporation device, e.g. an electroporation device forintradermal or intramuscular delivery. Suitably, a device as describedin U.S. Pat. No. 7,245,963B2 may be used, in particular a device asdefined by claims 1 to 68 of U.S. Pat. No. 7,245,963B2.

In embodiments where adenovirus DNA is used and comprised in thecomposition or vaccine or combination, thecomposition/vaccine/combination may be administered by intranasaladministration.

In embodiments, the nucleic acid as comprised in a composition orvaccine or combination as defined herein is provided in an amount ofabout 100 ng to about 500 ug, in an amount of about 1 ug to about 200ug, in an amount of about 1 ug to about 100 ug, in an amount of about 5ug to about 100 ug, preferably in an amount of about bug to about 50 ug,specifically, in an amount of about lug, 2 ug, 3 ug, 4 ug, 5 ug, 8 ug, 9ug,10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 16 ug 20 ug, 25 ug, 30 ug,35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80 ug, 85ug, 90 ug, 95 ug or 100 ug.

In some embodiments, the vaccine comprising the nucleic acid, or thecomposition comprising the nucleic acid is formulated in an effectiveamount to produce an antigen specific immune response in a subject. Insome embodiments, the effective amount of nucleic acid is a total doseof 1 ug to 200 ug, 1 ug to 100 ug, or 5 ug to 100 ug.

In embodiments where the nucleic acid is provided in a lipid-basedcarrier, e.g. an LNP, the amount of PEG-lipid as defined hereincomprised in one dose is lower than about 50 μg PEG lipid, preferablylower than about 45 μg PEG lipid, more preferably lower than about 40 μgPEG lipid.

Having a low amount of PEG lipid in one dose may reduce the risk ofadverse effects (e.g. allergies).

In particularly preferred embodiments, the amount of PEG-lipid comprisedin one dose is in a range from about 3.5 μg PEG lipid to about 35 μg PEGlipid.

In embodiments where the nucleic acid is provided in a lipid-basedcarrier, e.g. an LNP, the amount of cationic lipid as defined hereincomprised in one dose is lower than about 400 μg cationic lipid,preferably lower than about 350 μg cationic lipid, more preferably lowerthan about 300 μg cationic lipid.

Having a low amount of cationic lipid in one dose may reduce the risk ofadverse effects (e.g. fewer).

In particularly preferred embodiments, the amount of cationic-lipidcomprised in one dose is in a range from about 30 μg PEG lipid to about300 μg PEG lipid.

In one embodiment, the immunization protocol for the treatment orprophylaxis of a subject against coronavirus, preferably SARS-CoV-2coronavirus comprises one single doses of the composition or thevaccine.

In some embodiments, the effective amount is a dose of 1 ug administeredto the subject in one vaccination. In some embodiments, the effectiveamount is a dose of 2 ug administered to the subject in one vaccination.In some embodiments, the effective amount is a dose of 3 ug administeredto the subject in one vaccination. In some embodiments, the effectiveamount is a dose of 4 ug administered to the subject in one vaccination.In some embodiments, the effective amount is a dose of 5 ug administeredto the subject in one vaccination. 6 ug administered to the subject inone vaccination. In some embodiments, the effective amount is a dose of7 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 8 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 9 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 10 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 11 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 12 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 13 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 14 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 16 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 20 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 25 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 30 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 40 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 50 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 100 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 200 ug administered tothe subject in one vaccination. A “dose” in that context relates to theeffective amount of nucleic acid, preferably mRNA as defined herein.

In preferred embodiments, the immunization protocol for the treatment orprophylaxis of a coronavirus, preferably a SARS-CoV-2 coronavirusinfection comprises a series of single doses or dosages of thecomposition or the vaccine. A single dosage, as used herein, refers tothe initial/first dose, a second dose or any further doses,respectively, which are preferably administered in order to “boost” theimmune reaction.

In some embodiments, the effective amount is a dose of 1 ug administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 2 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 3 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 4 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 5 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 6 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 7 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 8 ug administered tothe subject a total of two times. In some embodiments, the effectiveamount is a dose of 9 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 10 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 11 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 12 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 13 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 14 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 16 ug administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 20 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 25 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 30 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 40 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 50 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 100 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 200 ug administeredto the subject a total of two times. A “dose” in that context relates tothe effective amount of nucleic acid, preferably mRNA as defined herein.

In preferred embodiments, the vaccine/composition/combination immunizesthe subject against a coronavirus, preferably against a SARS-CoV-2coronavirus infection (upon administration as defined herein) for atleast 1 year, preferably at least 2 years. In preferred embodiments, thevaccine/composition/combination immunizes the subject against acoronavirus, preferably against a SARS-CoV-2 coronavirus for more than 2years, more preferably for more than 3 years, even more preferably formore than 4 years, even more preferably for more than 5-10 years.

Method of Treatment and Use, Diagnostic Method and Use:

In another aspect, the present invention relates to a method of treatingor preventing a disorder.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, the kit or kit of parts ofthe fifth aspect, the combination of the sixth aspect, or medical usesmay likewise be read on and be understood as suitable embodiments ofmethods of treatments as provided herein. Furthermore, specific featuresand embodiments relating to method of treatments as provided herein mayalso apply for medical uses of the invention.

Preventing (Inhibiting) or treating a disease, in particular acoronavirus infection relates to inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as a coronavirus infection. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop. The term“ameliorating”, with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Inhibitinga disease can include preventing or reducing the risk of the disease,such as preventing or reducing the risk of viral infection. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

In preferred embodiments, the present invention relates to a method oftreating or preventing a disorder, wherein the method comprises applyingor administering to a subject in need thereof at least one nucleic acidof the first aspect, the composition of the second aspect, thepolypeptide of the third aspect, the vaccine of the fourth aspect, orthe kit or kit of parts of the fifth aspect, or the combination of thesixth aspect.

In preferred embodiments, the disorder is an infection with acoronavirus, or a disorder related to such infections, in particular aninfection with SARS-CoV-2 coronavirus, or a disorder related to suchinfections, e.g. COVID-19.

In preferred embodiments, the present invention relates to a method oftreating or preventing a disorder as defined above, wherein the methodcomprises applying or administering to a subject in need thereof atleast one nucleic acid of the first aspect, the composition of thesecond aspect, the polypeptide of the third aspect, the vaccine of thefourth aspect, or the kit or kit of parts of the fifth aspect, or thecombination of the sixth aspect, wherein the subject in need ispreferably a mammalian subject.

In certain embodiments, a method of treating or preventing disease byapplying or administering to a subject in need thereof at least onenucleic acid of the first aspect, the composition of the second aspect,the polypeptide of the third aspect, the vaccine of the fourth aspect,or the kit or kit of parts of the fifth aspect or the combination of thesixth aspect, is further defined as a method of reducing disease burdenin the subject. For example, the method preferably reduces the severityand/or duration of one or more symptom of COVID-19 disease. In someaspects, a method reduces the probability that a subject will requirehospital admission, intensive care unit admission, treatment withsupplemental oxygen and/or treatment with a ventilator. In furtheraspects, the method reduces the probability that a subject will developa fever, breathing difficulties; loss of smell and/or loss of taste. Inpreferred aspects, the method reduces the probability that a subjectwill develop severe or moderate COVID-19 disease. In certain aspects, amethod of the embodiments prevents severe or moderate COVID-19 diseasein the subject between about 2 weeks and 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 1 year or 2 years after the subject isadministered a composition of the embodiments. In preferred aspects, amethod of the embodiments prevents symptomatic COVID-19 disease. Infurther aspects, a method of the embodiment prevents detectable levelsof SARS-CoV-2 nucleic acid in the subject between about 2 weeks and 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or 2years after the subject is administered a composition of theembodiments. In further aspects, a method of the embodiments is definedas a method for providing protective immunity to a coronavirus infection(e.g., SARS-CoV-2 infection) in the subject. In still further aspects, amethod of the embodiments prevents moderate and severe COVID-19 diseasein at least 80%, 85%, 90% or 95% of treated subjects. In yet furtheraspects, a method of the embodiments prevents moderate and severeCOVID-19 disease in at least 80%, 85%, 90% or 95% of treated subjectsfrom about 2 weeks to about 1 year after administering the second orsubsequent immunogenic composition (e.g., a booster administration). Inyet further aspects, a method of the embodiments prevents moderate andsevere COVID-19 disease in at least 80%, 85%, 90% or 95% of treatedsubjects from about 2 weeks to about 3 month, 6 months, 9 months, 1year, 1.5 years, 2 years or 3 years after administering the second orsubsequent composition.

In a further aspects, a method of the embodiments comprises (i)obtaining a composition (e.g., a vaccine composition) of theembodiments, wherein the composition is lyophilized; (ii) solubilizingthe lyophilized composition in a pharmaceutically acceptable liquidcarrier to produce a liquid composition; and (iii) administering aneffective amount of the liquid composition to the subject. In someaspects, the lyophilized composition comprises less than about 10% watercontent. For example, the lyophilized composition can preferablycomprise about 0.1% to about 10%, 0.5% to 7.5% or 0.5% to 5.0% water.

In still further aspects, a method of the embodiments comprisesadministering a vaccine composition comprising at least two differentmRNAs, each mRNA encoding a different SARS-CoV-2 spike polypeptide thatare each at least about 95% identical to SEQ ID NO: 10 (e.g., in complexwith a LNP) to a subject. In further aspects, such a method provides asufficient immune response in the subject to protect the subject fromsevere COVID-19 disease for at least about 6 months. For example, insome aspects, the subject is protected from severe COVID-19 disease forabout 6 months to about 1 year, 1.5 years, 2 years, 2.5 years, 3 years,4 years or 5 years. Thus, in some aspects, a method of the embodimentsprovides a single dose vaccine composition that can provide prolonged(e.g., greater than 6 months of) protection from severe disease to asubject.

As used herein severe COVID-19 disease is defined as a subjectexperiencing one or more of the following:

Clinical signs at rest indicative of severe systemic illness(respiratory rate 30 breaths per minute, heart rate≥125 per minute,SpO2≤93% on room air at sea level or PaO2/FIO2<300 mm Hg (adjustedaccording to altitude))

Respiratory failure (defined as needing high flow-oxygen, noninvasiveventilation, mechanical ventilation or ECMO)

Evidence of shock (SBP<90 mm Hg, DBP<60 mmHg, or requiring vasopressors)

Significant renal, hepatic, or neurologic dysfunction

Admission to ICU

Death

As used herein moderate COVID-19 disease is defined as a subjectexperiencing one or more of the following:

Shortness of breath or difficulty breathing

Respiratory rate≥20 breaths per minute

Abnormal SpO2 but still >93% on room air at sea level (adjustedaccording to altitude)

Clinical or radiographic evidence of lower respiratory tract disease

Radiologic evidence of deep vein thrombosis (DVT)

As used herein mild COVID-19 disease is defined as a subjectexperiencing all of the following:

Symptomatic AND

No shortness of breath or difficulty breathing AND

No hypoxemia (adjusted according to altitude) AND

Does not meet the case definition of moderate or severe COVID-19 disease

In particularly preferred embodiments, the subject in need is amammalian subject, preferably a human subject, e.g. newborn, pregnant,immunocompromised, and/or elderly. In some embodiments, the subjectbetween the ages of 6 months and 100 years, 6 months and 80 years, 1year and 80 years, 1 year and 70 years, 2 years and 80 years or 2 yearsand 60 years. In other embodiments the subject is a newborn or infant ofan age of not more than 3 years, of not more than 2 years, of not morethan 1.5 years, of not more than 1 year (12 months), of not more than 9months, 6 months or 3 months. In certain embodiments, the human subjectis an elderly human subject. In some other embodiments the subject is anelderly subject of an age of at least 50, 60, 65, or 70 years. Infurther aspects, a subject for treatment according to the embodiments is61 years of age or older. In still further aspects, the subject is 18years old to 60 years old.

In further embodiments, the mammalian subject is a human subject is 60years of age or less. In certain embodiments the human subject is humansubject is 55, 50, 45 or 40 years of age or less. Thus, in someembodiments, is the human subject is between about 12 and 60; 12 and 55;12 and 50; 12 and 45; or 12 and 40 years of age. In further embodimentsthe human subject is between about 18 and 60; 18 and 55; 18 and 50; 18and 45; or 18 and 40 years of age. In some embodiments the human subjectis 18 to 50 or 18 to 40 years of age.

In certain embodiments, a subject for treatment according to theembodiments is a pregnant subject, such a pregnant human. In someaspects, the subject has been pregnant for more than about one month,two months, three months, four months, five months, six months, sevenmonths or eight months.

In certain aspects, a subject for treatment according to the embodimentshas native American, African, Asian or European heritage. In someaspects, the subject has at least about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% native American,African, Asian or European heritage. In certain aspects, the subject hasnative American heritage, such as at least about 10%, 25% or 50% nativeAmerican heritage. In further aspects, the subject is an elderly subjecthaving native American heritage, e.g., a subject who is at least 55, 60,65 or 70 years of age.

In further aspects, a subject for treatment according to the embodimentshas a disease or is immune compromised. In some aspects, the subject hasliver disease, kidney disease diabetes, hypertension, heart disease orlung disease. In further aspects, a subject for treatment according tothe embodiments is a subject with history of allergic reaction, such asubject having food allergies. In some aspect, the subject has had aprevious allergic reaction to a vaccine, such as an anaphylacticreaction. In still further aspects, a subject for treatment according tothe methods is a subject having detectable anti-PEG antibodies, such asdetectable anti-PEG IgE in the serum.

In further aspects, a subject for treatment according to the embodimentshas at least one co-morbidity selected from:

(i) Chronic kidney disease: Kidney function will be ascertained from theserum creatinine measurement within the last 3-6 months, converted intoestimated glomerular filtration rate (eGFR) using the Chronic KidneyDisease Epidemiology Collaboration (CKD-EPI) equation, with impairedkidney function defined as eGFR<60 mL/min/1.73 m².

Mild chronic kidney disease is defined as an eGFR between 60-89mL/min/1.73 m².

Moderate chronic kidney disease is defined as an eGFR between 31-59mL/min/1.73 m² with stable therapy and good maintenance over at least 6months (modified from Clinical Practice Clinical Guidelines for ChronicKidney Disease: Am J Kidney Dis, 2002).

(ii) COPD (including emphysema and chronic bronchitis).

Mild COPD with or without cough or sputum production is defined asforced expiratory volume in 1 second/forced vital capacity(FEV1/FVC)<0.7 and FEV1≥80% predicted.

Moderate COPD with or without cough or sputum production is defined asFEV1/FVC<0.7 and FEV1≥50%, but <80% predicted with stable treatment(GOLD Criteria for COPD severity).

(iii) Obesity with body mass index (BMI) of >32 kg/m²—any extreme morbidobesity will also be included.

(iv) Chronic cardiovascular conditions (heart failure, coronary arterydisease, cardiomyopathies, arterial hypertension), including thefollowing:

Class I heart failure with potential high risk for developing heartfailure in future with no functional or structural heart disorder.

Class II heart failure: subjects with cardiac disease resulting inslight limitation of physical activity. Comfortable at rest.

Ordinary physical activity results in fatigue, palpitation, dyspnea, oranginal pain.

Class III heart failure with marked limitation of physical activity, butcomfortable at rest but less than ordinary activity results in symptoms.

A structural heart disorder without symptoms at any stage.

Mild left ventricular systolic or diastolic dysfunction, usually withnot much produced clinical signs.

Moderate left ventricular failure with exertional dyspnea or orthopneaor paroxysmal nocturnal dyspnea as per New York Heart Association(NYHA), stable with medication (Class II-III).

Coronary artery disease of 2 and above metabolic equivalent threshold(MET) up to moderate, stable with medication. (MET is defined as theamount of oxygen consumed while sitting at rest and is equal to 3.5 mlO₂ per kg body weight×min; 4 Normal, can climb a flight of stairs orwalk up a hill and can participate in other strenuous activities; 1 cantake care of him/herself and may not maintain themselves and getsconstraints on exertion.)

Cardiomyopathies of non-infective and metabolic origin of 2-3 MET withmedication.

Stage 1 hypertension or Stage 2 hypertension stable and controlled withmedications.

(v) Chronic HIV infection with stable aviraemia (<50 copies/mL) and CD4count>350/mL as documented by blood samples taken within 12 monthsbefore enrolment. (Viral load<50 copies/mL with transient changes of50-350 copies/mL is allowed.)

(vi) Type 2 diabetes mellitus, either controlled with medication[hemoglobin A1c (HbA1c)<58 mmol/mol (7.45%)] or uncontrolled with recentHbA1c of >58 mmol/mol (7.45%); [(HbA1c in %−2.15)×10.929=HbA1c inmmol/mol]; in uncontrolled DM HbA1c should be within <10% variation andshould not have any history of diabetic ketoacidosis or episode ofsevere symptomatic hypoglycemia within prior 3 months.

(vii) Subjects who underwent renal transplant at least a year ago understable conditions for at least 6 months with medications, categorized aslow risk of rejection.

In still further aspects, a subject for treatment according to theembodiments has not been treated with an immunosuppressant drug for morethan 14 days in the last 6 months. In some aspects, a subject fortreatment according to the embodiments has not received a live vaccinefor at least 28 days prior to the administration and/or has not receivedan inactivated vaccine for at least 14 days prior to the administration.In further aspects a subject for treatment according to the embodimentshas NOT:

Had virologically-confirmed COVID-19 illness;

For females: experienced pregnancy or lactation with-in a month prior toadministration of the composition of the embodiments;

had treatment with an investigational or non-registered product (e.g.,vaccine or drug) within 28 days preceding the administration of thecomposition of the embodiments;

received a licensed vaccines within 28 days (for live vaccines) or 14days (for inactivated vaccines) prior to the administration of thecomposition of the embodiments;

been previously or concurrently treated with any investigationalSARS-CoV-2 vaccine or another coronavirus (SARS-CoV, MERS-CoV) vaccine;

been treated with immunosuppressants or other immune-modifying drugs(e.g., corticosteroids, biologicals and methotrexate) for >14 days totalwithin 6 months preceding the administration of the composition of theembodiments;

had any medically diagnosed or suspected immunosuppressive orimmunodeficient condition based on medical history and physicalexamination including known infection with human immunodeficiency virus(HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV); currentdiagnosis of or treatment for cancer including leukemia, lymphoma,Hodgkin disease, multiple myeloma, or generalized malignancy; chronicrenal failure or nephrotic syndrome; and receipt of an organ or bonemarrow transplant.

had a history of angioedema (hereditary or idiopathic), or history ofany anaphylactic reaction or pIMD.

a history of allergy to any component of CVnCoV vaccine.

been administered of immunoglobulins or any blood products within 3months prior to the administration of the composition of theembodiments;

experienced a significant acute or chronic medical or psychiatricillness; and/or

experienced severe and/or uncontrolled cardiovascular disease,gastrointestinal disease, liver disease, renal disease, respiratorydisease, endocrine disorder, and neurological and psychiatric illnesses.

In certain aspects, a subject for treatment according to the methods ofthe embodiments does not have any potential immune-mediated disease(pIMD). In further aspects, a treatment method of the embodiments doesnot induce any pIMD in a treated subject. As used herein pIMDs aredefined as Celiac disease; Crohn's disease; Ulcerative colitis;Ulcerative proctitis; Autoimmune cholangitis; Autoimmune hepatitis;Primary biliary cirrhosis; Primary sclerosing cholangitis; Addison'sdisease; Autoimmune thyroiditis (including Hashimoto thyroiditis;Diabetes mellitus type I; Grave's or Basedow's disease; Antisynthetasesyndrome; Dermatomyositis; Juvenile chronic arthritis (including Still'sdisease); Mixed connective tissue disorder; Polymyalgia rheumatic;Polymyositis; Psoriatic arthropathy; Relapsing polychondritis;Rheumatoid arthritis; Scleroderma, (e.g., including diffuse systemicform and CREST syndrome); Spondyloarthritis, (e.g., including ankylosingspondylitis, reactive arthritis (Reiter's Syndrome) and undifferentiatedspondyloarthritis); Systemic lupus erythematosus; Systemic sclerosis;Acute disseminated encephalomyelitis, (including site specific variants(e.g., non-infectious encephalitis, encephalomyelitis, myelitis,myeloradiculomyelitis)); Cranial nerve disorders,(e.g., includingparalyses/paresis (e.g., Bell's palsy)); Guillain-Barré syndrome, (e.g.,including Miller Fisher syndrome and other variants); Immune-mediatedperipheral neuropathies, Parsonage-Turner syndrome and plexopathies,(e.g., including chronic inflammatory demyelinating polyneuropathy,multifocal motor neuropathy, and polyneuropathies associated withmonoclonal gammopathy); Multiple sclerosis; Narcolepsy; Optic neuritis;Transverse Myelitis; Alopecia areata; Autoimmune bullous skin diseases,including pemphigus, pemphigoid and dermatitis herpetiformis; Cutaneouslupus erythematosus; Erythema nodosum; Morphoea; Lichen planus;Psoriasis; Sweet's syndrome; Vitiligo; Large vessels vasculitis (e.g.,including: giant cell arteritis such as Takayasu's arteritis andtemporal arteritis); Medium sized and/or small vessels vasculitis (e.g.,including: polyarteritis nodosa, Kawasaki's disease, microscopicpolyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome (allergicgranulomatous angiitis), Buerger's disease thromboangiitis obliterans,necrotizing vasculitis and anti-neutrophil cytoplasmic antibody (ANCA)positive vasculitis (type unspecified), Henoch-Schonlein purpura,Behcet's syndrome, leukocytoclastic vasculitis); Antiphospholipidsyndrome; Autoimmune hemolytic anemia; Autoimmune glomerulonephritis(including IgA nephropathy, glomerulonephritis rapidly progressive,membranous glomerulonephritis, membranoproliferative glomerulonephritis,and mesangioproliferative glomerulonephritis); Autoimmunemyocarditis/cardiomyopathy; Autoimmune thrombocytopenia; Goodpasturesyndrome; Idiopathic pulmonary fibrosis; Pernicious anemia; Raynaudvsphenomenon; Sarcoidosis; Sjögren's syndrome; Stevens-Johnson syndrome;Uveitis).

In certain aspects, a vaccination method of the embodiments does notresult in a subject experiencing any adverse events of special interest(AESIs). As used herein AESIs are defined as a pIMD listed above;Anaphylaxis; Vasculitides; Enhanced disease following immunization;Multisystem inflammatory syndrome in children; Acute RespiratoryDistress Syndrome; COVID-19 disease; Acute cardiac injury;Microangiopathy; Heart failure and cardiogenic shock; Stresscardiomyopathy; Coronary artery disease; Arrhythmia; Myocarditis,pericarditis; Thrombocytopenia; Deep vein thrombosis; Pulmonary embolus;Cerebrovascular stroke; Limb ischemia; Hemorrhagic disease; Acute kidneyinjury; Liver injury; Generalized convulsion; Guillain-Barré Syndrome;Acute disseminated encephalomyelitis; Anosmia, ageusia;Meningoencephalitis; Chilblain-like lesions; Single organ cutaneousvasculitis; Erythema multiforme; Serious local/systemic AR followingimmunization

In particular, such the method of treatment may comprise the steps of:

-   -   a) providing at least one nucleic acid (e.g. DNA or RNA),        preferably at least one RNA of the first aspect, at least one        composition of the second aspect, at least one polypeptide of        the third aspect, at least one vaccine of the fourth aspect, or        the kit or kit of parts of the fifth aspect;    -   b) applying or administering said nucleic acid, composition,        polypeptide, vaccine, or kit or kit of parts to a subject as a        first dose    -   c) optionally, applying or administering said nucleic acid,        composition, polypeptide, vaccine, or kit or kit of parts to a        subject as a second dose or a further dose, preferably at least        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, months after the first dose.

The first dosage, as used herein, refers to the initial/first dose, asecond dose or any further doses, respectively, which are preferablyadministered in order to “boost” the immune reaction. In certainaspects, the vaccine/composition is administered to a subject one, twothree, four or more times. In some aspects, the vaccine/composition isadministered to the subject at least first and a second time (e.g., aprime and boost). I some aspects, the send administration is at least 10days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days or 56 daysafter the first administration. In some aspects, the time between thefirst administration and the second administration is between about 7days and about 56 days; about 14 days and about 56 days; about 21 daysand about 56 days; or about 28 days and about 56 days. In furtheraspects, the vaccine/composition is administered to a subject three ormore times. In certain aspects, there is at least 10 days, 14 days, 21days, 28 days, 35 days, 42 days, 49 days or 56 days between eachadministration of the vaccine/composition.

In some aspects, a subject for treatment according to the embodimentswas previously infected with SARS-CoV-2 or was previously treated withat least a first SARS-CoV-2 vaccine composition. In some aspects, thesubject was treated with one, two, three or more doses of a firstSARS-CoV-2 vaccine composition. In some aspects, the composition of theembodiments used to treat a subject is a different type of vaccinecomposition than the composition previously used to treat the subject.In some aspects, the subject was previously treated with a mRNA vaccine,such as BNT162 or mRNA-1273. In further aspects, the subject waspreviously treated with a protein subunit vaccine, such as spike proteinbased vaccine, e.g., NVX-CoV2373 or COVAX. In certain preferred aspects,protein subunit vaccine compositions comprise an adjuvant. In furtheraspects, the subject was previously treated with a viral vector vaccine,such as an adenovirus vector based vaccine, e.g., ADZ1222 orAd26.COV-2.S. In still further aspects, the subject was previouslytreated with an inactivated virus vaccine to SARS-CoV-2 such asCoronaVac, BBIBP-CorV or BBV152. In further aspects, a subjectpreviously treated with a vaccine composition has detectable SARS-CoV-2binding antibodies, such as SARS-CoV-2 S protein-binding antibodies orSARS-CoV-2 N protein-binding antibodies. In further aspects, a subjectfor treatment according the embodiments was treated with a firstSARS-CoV-2 vaccine composition at least about 3 month, 6 months, 9months, 1 year, 1.5 years, 2 years or 3 years ago. In still furtheraspects, a subject for treatment according the embodiments was treatedwith a first SARS-CoV-2 vaccine composition between about 3 months and 2years ago or between about 6 months and 2 years ago. In some aspects, asubjects treated with a further vaccine composition of the embodimentsare protected from moderate and severe COVID-19 disease in at least 80%,85%, 90% or 95% of treated subjects. For example, the treated subjectscan be protected from moderate and severe COVID-19 disease in at least80%, 85%, 90% or 95% of treated subjects from about 2 weeks to about 1year after administration of the further composition. In still furtheraspects, administering the further vaccine composition of theembodiments prevents moderate and severe COVID-19 disease in at least80%, 85%, 90% or 95% of treated subjects from about 2 weeks to about 3month, 6 months, 9 months, 1 year, 1.5 years, 2 years or 3 years aftersaid administration. Examples of such combination vaccination strategiesare shown below:

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 mRNA vaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 protein subunitvaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 viral vectorvaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 inactivated virusvaccine

Dose 1 protein subunit vaccine—T1—dose 2 protein subunit vaccine—T2—dose3 mRNA vaccine

Dose 1 inactivated virus vaccine—T1—dose 2 inactivated virusvaccine—T2—dose 3 mRNA vaccine

Dose 1 viral vector vaccine—T1—dose 2 viral vector vaccine—T2—dose 3mRNA vaccine

Dose 1 viral vector vaccine—T2—dose 2 mRNA vaccine

Dose 1 protein subunit vaccine—T2—dose 2 mRNA vaccine

Dose 1 inactivated virus vaccine—T2—dose 2 mRNA vaccine

Dose 1 mRNA vaccine—T2—dose 2 mRNA vaccine

In the examples, above time period 1 (T1) is typically 2 to 6 weeks,preferably 3 to 4 weeks. Time period 2 (T2) is in some cases, about 3months, 6 months, 9 months, 1 year, 1.5 years, 2 years or three years.

In some aspects, a method of the embodiments comprises administeringmultiple doses of a vaccine composition to a subject. In a furtheraspect, there is provided a method of reducing reactogenicity of aSARS-CoV-2 booster vaccine composition. In some aspects, after aninitial vaccination, subject exhibiting a high level of reactogenicityare administered a booster vaccine that is different from the initialvaccine composition. For example, in some aspects, the initial vaccineis BNT162 or mRNA-1273 and the booster vaccine is a mRNA vaccinecomposition of the embodiments. In some aspects, a booster vaccinecomposition for a subject with high reactogenicity is selected basedhaving a lower concentration of PEG or PEG-conjugate compared to thepreviously administered vaccine composition. In some aspects, a boostervaccine composition for a subject with high reactogenicity is selectedbased on a lower concentration of mRNA or LNP compared to the previouslyadministered vaccine composition.

In certain aspects, a subject for treatment according to the embodimentsis administered a vaccine composition as booster vaccine and haspreviously been treated with one or more administrations of acoronavirus vaccine composition. In certain aspects, the subject beingtreated with a booster vaccine previously was treated with a vaccinecomposition that included a spike protein antigen or a nucleic acidmolecule encoding a spike protein antigen. In some aspects, the subjectselected for treatment with the booster vaccine was previouslyadministered a vaccine composition comprising, or encoding, a spikeprotein having a different amino acid sequence than the spike protein ofthe booster vaccine. In certain aspects, the previously administeredvaccine composition comprised, or encoded, a spike (e.g., a SARS-CoV-2spike) protein having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid differences relative to the booster vaccine composition. In certainaspects, the booster vaccine composition comprises a RNA encoding aspike protein having about 1 to 50; about 3 to 30; about 5 to 30 orabout 10 to 25 amino acid differences relative to the previouslyadministered vaccine composition. In still further aspects, the boostervaccine composition comprises RNA encoding 2, 3, 4 or more distinctspike proteins with different amino acid sequences.

In further aspects, methods of the embodiments comprise administering 2or more booster vaccine compositions to a subject, wherein each boostervaccine composition comprises RNA encoding a distinct spike protein withdifferent amino acid sequences. In some aspects, such distinct boostervaccine compositions are administered essentially simultaneously or lessthan about 10 minutes, 20 minutes, 30 minutes, 1 hour or 2 hours apart.In some aspects, distinct booster vaccine compositions are administeredto the same site, such as intramuscular injections to the same arm ofthe subject. In further aspects, distinct booster vaccine compositionsare administered to different sites, such as intramuscular injections todifferent arms or to one or both arms and one more leg muscles.

In certain aspects, a method of the embodiments is further defined as amethod of stimulating an antibody or CD8+ T-cell response in a subject.In some aspects, the method is defined as a method of stimulating aneutralizing antibody response in a subject. In further aspects, themethod is defined as a method of stimulating a protective immuneresponse in a subject. In yet further aspects, the method is defined asa method of stimulating TH2 directed immune response in a subject.

In further aspects, administration of a vaccine/composition/combinationof the embodiments stimulates an antibody response that produces betweenabout 10 and about 500 coronavirus spike protein-binding antibodies forevery coronavirus neutralizing antibody in the subject. For example, theadministration can stimulate an antibody response that produces no morethan about 200 spike protein-binding antibodies for every coronavirusneutralizing antibody. In further aspects, the administration stimulatesan antibody response that produces between about 10 and about 300; about20 and about 300; about 20 and about 200; about 30 and about 100; orabout 30 and about 80 coronavirus spike protein-binding antibodies forevery coronavirus neutralizing antibody. In still further aspects,administration of composition of the embodiments stimulates an antibodyresponse in a subject that includes a ratio of spike protein-bindingantibodies to coronavirus neutralizing antibodies that is with 20%, 15%,10% or 5% of the ratio of spike protein-binding antibodies tocoronavirus neutralizing antibodies found in average convalescentpatient serum (from a subject who has recovered from coronavirusinfection).

In yet further aspects, administration of avaccine/composition/combination of the embodiments stimulates anantibody response that produces between about 1 and about 500coronavirus spike protein receptor binding domain (RBD)-bindingantibodies for every coronavirus neutralizing antibody in the subject.In further aspects, the administration stimulates an antibody responsethat produces no more than about 50 spike protein RBD-binding antibodiesfor every coronavirus neutralizing antibody. In still further aspects,administration stimulates an antibody response that produces betweenabout 1 and about 200; about 2 and about 100; about 3 and about 200;about 5 and about 100; about 5 and about 50; or about 5 and about 20spike protein RBD-binding antibodies for every coronavirus neutralizingantibody. In still further aspects, administration of composition of theembodiments stimulates an antibody response in a subject that includes aratio of spike protein RBD-binding antibodies to coronavirusneutralizing antibodies that is with 20%, 15%, 10% or 5% of the ratio ofspike protein RBD-binding antibodies to coronavirus neutralizingantibodies found in average convalescent patient serum (from a subjectwho has recovered from coronavirus infection).

In still further aspects, administration of avaccine/composition/combination of the embodiments induces essentiallyno increase in IL-4, IL-13, TNF and/or IL-1β in the subject. In furtheraspects, the administration of a vaccine/composition of the embodimentsinduces essentially no increase in serum IL-4, IL-13, TNF and/or IL-1βin the subject. In some aspects, the administration of avaccine/composition of the embodiments induces essentially no increasein IL-4, IL-13, TNF and/or IL-1β at the injection site (e.g., anintramuscular injection site) in the subject. In still further aspects,a method of the embodiments comprises administration of avaccine/composition of the embodiments to a human subject having adisease. In certain aspects, the subject has cardiovascular disease,kidney disease, lung disease or an autoimmune disease. In some aspects,a vaccine/composition of the embodiments is administered to a subjectwho is receiving anti-coagulation therapy.

In still further aspects, administering avaccine/composition/combination of the embodiments to human subjectsresults in no more than 20%, 15%, 10% 7.5% or 5% of the subjectsexperiencing a Grade 3 local adverse event (see Table 3a below). Forexample, in some aspects, no more than 10% of subjects experience aGrade 3 local adverse event after a first or a second dose of thecomposition. In preferred aspects, administering a composition of theembodiments to human subjects results in no more than 40%, 30%, 25%,20%, 15%, 10%, 7.5% or 5% of the subjects experiencing a Grade 2 ofhigher local adverse event. For example, in some aspects, no more than30% of subjects experience a Grade 2 or higher local adverse event aftera first or a second dose of the composition. In some aspects,administering a composition of the embodiments to human subjects resultsin no more than 10% of the subjects experiencing Grade 3 pain, redness,swelling and/or itching at the injection site

In further aspects, administering a vaccine/composition/combination ofthe embodiments to human subjects results in no more than 30%, 25%, 20%,15%, 10% or 5% of the subjects experiencing a Grade 3 systemic adverseevent (see Table B below). For example, in some aspects, no more than25% of subjects experience a Grade 3 systemic adverse event after afirst dose of the composition. In some aspects, no more than 40% ofsubjects experience a Grade 3 systemic adverse event after a second doseof the composition. In some aspects, administering a composition of theembodiments to human subjects results in no more than 30%, 25%, 20%,15%, 10% or 5% of the subjects experiencing Grade 3 fever, headache,fatigue, chills, myalgia, arthralgia, nausea and/or diarrhea.

TABLE 3a Intensity Grading* for Solicited Local Adverse Events AE GradeDefinition Pain at 0 Absent Injection 1 Does not interfere with activitySite 2 Interferes with activity and/or repeated use of non-narcotic painreliever >24 hours 3 Prevents daily activity and/or repeated use ofnarcotic pain reliever Redness 0 <2.5 cm 1 2.5-5 cm 2 5.1-10 cm 3 >10 cmSwelling 0 <2.5 cm 1 2.5-5 cm and does not interfere with activity 25.1-10 cm or interferes with activity 3 >10 cm or prevents dailyactivity Itching 0 Absent 1 Mild, no interference with normal activity 2Moderate, some interference with normal activity 3 Significant, preventsnormal activity

TABLE 3b Intensity Grading* for Solicited Systemic Adverse EventsAdverse Event Grade Definition Fever 0 <38° C. 1 ≥38.0-38.4° C. 2≥38.5-38.9° C. 3 ≥39° C. Headache 0 Absent 1 Mild, no interference withnormal activity 2 Moderate, some interference with normal activityand/or repeated use of non-narcotic pain reliever >24 hours 3Significant; any use of narcotic pain reliever and/or prevents dailyactivity Fatigue 0 Absent 1 Mild, no interference with normal activity 2Moderate, some interference with normal activity 3 Significant, preventsnormal activity Chills 0 Absent 1 Mild, no interference with normalactivity 2 Moderate, some interference with normal activity 3Significant, prevents normal activity Myalgia 0 Absent 1 Mild, nointerference with normal activity 2 Moderate, some interference withnormal activity 3 Significant, prevents normal activity Arthralgia 0Absent 1 Mild, no interference with normal activity 2 Moderate, someinterference with normal activity 3 Significant, prevents normalactivity Nausea/ 0 Absent Vomiting 1 Mild, no interference with activityand/or 1-2 episodes/24 hours 2 Moderate, some interference with activityand/or >2 episodes/24 hours 3 Significant, prevents daily activity,requires outpatient IV hydration Diarrhea 0 Absent 1 2-3 loose stoolsover 24 hours 2 4-5 stools over 24 hours 3 6 or more watery stools over24 hours or requires outpatient IV hydration *FDA toxicity grading scale(US Department of Health and Human Services. Food and DrugAdministration (FDA). Guidance for Industry. Toxicity Grading Scale forHealthy Adult and Adolescent Volunteers Enrolled in Preventive VaccineClinical Trials. 2007. On the world wide web atfda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatorylnformation/Guidances/Vaccines/ucm091977.pdf;Accessed at: March 2019, incorporated herein by reference); IV =Intravenous.

According to a further aspect, the present invention also provides amethod for expression of at least one polypeptide comprising at leastone peptide or protein derived from a coronavirus, or a fragment orvariant thereof, wherein the method preferably comprises the followingsteps:

-   -   a) providing at least one nucleic acid of the first aspect or at        least one composition of the second aspect; and    -   b) applying or administering said nucleic acid or composition to        an expression system (cells), a tissue, an organism. A suitable        cell for expressing a polypeptide (that is encoded by the        nucleic acid of the invention) may be a Drosophila S2 insect        cell line.

The method for expression may be applied for laboratory, for research,for diagnostic, for commercial production of peptides or proteins and/orfor therapeutic purposes. The method may furthermore be carried out inthe context of the treatment of a specific disease, particularly in thetreatment of infectious diseases, particularly coronavirus infections,preferably SARS-CoV-2 coronavirus infections and the disease COVID-19.

Likewise, according to another aspect, the present invention alsoprovides the use of the nucleic acid, the composition, the polypeptide,the vaccine, or the kit or kit of parts preferably for diagnostic ortherapeutic purposes, e.g. for expression of an encoded coronavirusantigenic peptide or protein.

In specific embodiments, applying or administering said nucleic acid,polypeptide, composition, vaccine, combination to a tissue or anorganism may be followed by e.g. a step of obtaining induced coronavirusantibodies e.g. SARS-CoV-2 coronavirus specific (monoclonal) antibodiesor a step of obtaining generated SARS-CoV-2 coronavirus proteinconstructs (S protein).

The use may be applied for a (diagnostic) laboratory, for research, fordiagnostics, for commercial production of peptides, proteins, orSARS-CoV-2 coronavirus antibodies and/or for therapeutic purposes. Theuse may be carried out in vitro, in vivo or ex vivo. The use mayfurthermore be carried out in the context of the treatment of a specificdisease, particularly in the treatment of a coronavirus infection (e.g.COVID-19) or a related disorder.

According to a further aspect, the present invention also provides amethod of manufacturing a composition or a vaccine, comprising the stepsof:

-   -   a) RNA in vitro transcription step using a DNA template in the        presence of a cap analogue to obtain capped mRNA, preferably        having a nucleic acid sequence as provided in Table 2;    -   b) Purifying the obtained capped RNA of step a) using RP-HPLC,        and/or TFF, and/or Oligo(dT) purification and/or AEX, preferably        using RP-HPLC;    -   c) Providing a first liquid composition comprising the purified        capped RNA of step b);    -   d) Providing a second liquid composition comprising at least one        cationic lipid as defined herein, a neutral lipid as defined        herein, a steroid or steroid analogue as defined herein, and a        PEG-lipid as defined herein;    -   e) Introducing the first liquid composition and the second        liquid composition into at least one mixing means to allow the        formation of LNPs comprising capped RNA;    -   f) Purifying the obtained LNPs comprising capped RNA;    -   g) optionally, lyophilizing the purified LNPs comprising capped        RNA.

Preferably, the mixing means of step e) is a T-piece connector or amicrofluidic mixing device. Preferably, the purifying step f) comprisesat least one step selected from precipitation step, dialysis step,filtration step, TFF step. Optionally, an enzymatic polyadenylation stepmay be performed after step a) or b). Optionally, further purificationsteps may be implemented to e.g. remove residual DNA, buffers, small RNAby-products etc. Optionally, RNA in vitro transcription is performed inthe absence of a cap analog, and an enzymatic capping step is performedafter RNA vitro transcription. Optionally, RNA in vitro transcription isperformed in the presence of at least one modified nucleotide as definedherein.

In embodiments, step a, preferably steps a-c, more preferably all stepsoutlined above (a-g) are performed in an automated device for RNA invitro transcription. Such a device may also be used to produce thecomposition or the vaccine (see aspects 2 and 3). Preferably, a deviceas described in WO2020/002598, in particular, a device as described inclaims 1 to 59 and/or 68 to 76 of WO2020/002598 (and FIGS. 1-18) maysuitably be used.

BRIEF DESCRIPTION OF LISTS AND TABLES

List 1a: Amino acid positions for substiutions deletions and/orinsertions

List 1b: Amino acid substiutions deletions or insertions

Table 1: Preferred coronavirus constructs (amino acid sequences andnucleic acid coding sequences)

Table 2a: RNA constructs suitable for a coronavirus vaccine

Table 2b: RNA constructs suitable for a coronavirus vaccine

Table 3a: Intensity Grading for Solicited Local Adverse Events

Table 3b: Intensity Grading for Solicited Systemic Adverse Events

Table 4: RNA constructs encoding different SARS-CoV-2 S antigen design(used in the Examples)

Table 5: Lipid-based carrier composition of the examples

Table 6: Vaccination regimen

Table 7: Median VNTs (day 42)

Table 8: Vaccination regimen (Example 3)

Table 9: Vaccination regimen (Example 4)

Table 10: Vaccination regimen (Example 5)

Table 11: Vaccination regimen (Example 6)

Table 12: Vaccination regimen (Example 7)

Table 13: Vaccination regimen (Example 8)

Table 14A: Vaccination regime (Example 9A)

Table 14B: Vaccination regimen (Example 9B)

Table 15: Vaccination regimen (Example 10)

Table 16: Vaccination regimen (Example 11)

Table 17: Vaccination regimen (Example 12)

Table 18: Vaccination regime (Example 13)

Table 19: Vaccination regime (Example 14)

EXAMPLES

The following examples illustrate various embodiments and aspects of theinvention. The present invention is not intended to in any way belimited in scope by the specific embodiments described herein. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods, which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become readily apparent to those skilled in the art from theforegoing description, accompanying figures and the examples below. Allsuch modifications fall within the scope of the appended claims.

Example 1 Preparation of DNA and RNA Constructs, Compositions, andVaccines

The present Example provides methods of obtaining the RNA of theinvention as well as methods of generating a composition or a vaccine ofthe invention.

1.1. Preparation of DNA and RNA Constructs:

DNA sequences encoding different SARS-CoV-2 S protein designs wereprepared and used for subsequent RNA in vitro transcription reactions.The DNA sequences were prepared by modifying the wild type or referenceencoding DNA sequences by introducing a G/C optimized or modified codingsequence (e.g., “cds opt1”) for stabilization and expressionoptimization. Sequences were introduced into a pUC derived DNA vector toproduce stabilizing 3′-UTR sequences and 5′-UTR sequences, additionallyhaving a stretch of adenosines (e.g. A64 or A100), and optionally ahistone-stem-loop (hSL) structure, and optionally a stretch of 30cytosines (e.g. C30) (see Table 4, for an overview of coronavirusantigen designs see List 1 or Table 1).

The obtained plasmid DNA constructs were transformed and propagated inbacteria using common protocols known in the art. The plasmid DNAconstructs were extracted, purified, and used for subsequent RNA invitro transcription (see section 1.2.).

Alternatively, DNA plasmids can be used as template forPCR-amplification (see section 1.3.).

1.2. RNA In Vitro Transcription from Plasmid DNA Templates:

DNA plasmids prepared according to section 1.1 were enzymaticallylinearized using a restriction enzyme and used for DNA dependent RNA invitro transcription using T7 RNA polymerase in the presence of anucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (e.g. m7GpppG,m7G(5′)ppp(5′)(2′OMeA)pG,m7G(5′)ppp(5′)(2′OMeG)pG), or3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG.) under suitable buffer conditions. Theobtained RNA constructs were purified using RP-HPLC (PureMessenger®,CureVac AG, Tübingen, Germany; WO2008/077592) and used for in vitro andin vivo experiments. DNA templates may also be generated using PCR. SuchPCR templates can be used for DNA dependent RNA in vitro transcriptionusing an RNA polymerase as outlined herein.

To obtain chemically modified mRNA, RNA in vitro transcription wasperformed in the presence of a modified nucleotide mixture comprisingN(1)-methylpseudouridine (m1ψ) or pseudouridine (ψ) instead of uracil.The obtained m1ψ or ψ chemically modified RNA was purified using RP-HPLC(PureMessenger®, CureVac AG, Tübingen, Germany; WO2008/077592) and usedfor further experiments.

Generation of Capped RNA Using Enzymatic Capping (Prophetic):

Some RNA constructs are in vitro transcribed in the absence of a capanalog. The cap-structure (cap0 or cap1) is then added enzymaticallyusing capping enzymes as commonly known in the art. In vitro transcribedRNA is capped using a capping kit to obtain cap0-RNA. cap0-RNA isadditionally modified using cap specific 2′-O-methyltransferase toobtain cap1-RNA. cap1-RNA is purified e.g. as explained above and usedfor further experiments.

RNA for clinical development is produced under current goodmanufacturing practice e.g. according to WO2016/180430, implementingvarious quality control steps on DNA and RNA level.

The RNA Constructs of the Examples:

The generated RNA sequences/constructs are provided in Table 4 with theencoded antigenic protein and the respective UTR elements indicatedtherein. If not indicated otherwise, the RNA sequences/constructs ofTable 4 were produced using RNA in vitro transcription in the presenceof a m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG; accordingly, the RNAsequences/constructs comprise a 5′ Cap1 structure. If not indicatedotherwise, the RNA sequences/constructs of Table 4 have been produced inthe absence of chemically modified nucleotides (e.g. pseudouridine (ψ)or N(1)-methylpseudouridine (m1ψ)).

1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates(Prophetic):

Purified PCR amplified DNA templates prepared according to paragraph 1.1is transcribed in vitro using DNA dependent T7 RNA polymerase in thepresence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog(m7GpppG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions.Alternatively, PCR amplified DNA is transcribed in vitro using DNAdependent T7 RNA polymerase in the presence of a modified nucleotidemixture (ATP, GTP, CTP, N1-methylpseudouridine (m1ψ) or pseudouridine(ψ) and cap analogue (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG orm7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. Some RNAconstructs are in vitro transcribed in the absence of a cap analog andthe cap-structure (cap0 or cap1) is added enzymatically using cappingenzymes as commonly known in the art. The obtained RNA is purified e.g.as explained above and used for further experiments. The obtained mRNAsare purified e.g. using RP-HPLC (PureMessenger®, CureVac AG, Tübingen,Germany; WO2008/077592) and used for in vitro and in vivo experiments.

TABLE 4 RNA constructs encoding different SARS-CoV-2 S antigen designsRNA ID Short name R9488, S R9492, R9487, S_stab_PP (K986P_V987P) R9491,R9709, R10159**, R10160, R10727**, R10820**, R10821** R10166, S_stab_PP(K986P_V987P_D614G) R10812**, R10813** R10811,S_stab_PP(K986P_V987P_D614G) R10814**, R10815** R10279 S_stab_PP(K986P_V987P_A222V_D614G) R10299 S_stab_PP (K986P_V987P_N439K_D614G)R10286 S_stab_PP (K986P_V987P_S477N_D614G)S_stab_PP(K986P_V987P_N501Y_D614G) R10275 S_stab_PP(K986P_V987P_H69del_V70del_D614G) R10283 S_stab_PP(K986P_V987P_Y453F_D614G) R10291 S_stab_PP (K986P_V987P_D614G_I692V)S_stab_PP(K986P_V987P_D614G_M1229I) R10295 S_stab_PP(K986P_V987P_H69del_V70del_A222V_(—) Y453F_S477N_D614G_I692V)S_stab_PP(K986P_V987P_H69del_V70del_Y453F_(—) D614G_I692V_M1229I)R10162** S_stab_PP (K986P_V987P) R10357 S_stab_PP(K986P_V987P_H69del_V70del_Y144del_N501Y_A570D_(—)D614G_P681H_T716I_S982A_D1118H) R10361 S_stab_PP(K986P_V987P_K417N_E484K_N501Y_D614G) R10384 S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_A243del_(—)L244del_R246I_K417N_E484K_N501Y_D614G_A701V) R10385 S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_K417T_(—)E484K_N501Y_D614G_H655Y_T1027I) R10410 S_stab_PP(K986P_V987P_H69del_V70del_Y144del_N501Y_A570D_(—)D614G_P681H_T716I_S982A_D1118H_E484K) R10452 S_stab_PP(K986P_V987P_L18F_D80A_D215G_L242del_A243del_(—)L244del_R246I_K417N_E484K_N501Y_D614G_A701V) R9515 S_stab_PP(K986P_V987P) R10385S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_(—)K417T_E484K_N501Y_D614G_H655Y_T1027I) R10616S_stab_PP(K986P_V987P_L18F_T20N_P26S_D138Y_R190S_(—)K417T_E484K_N501Y_D614G_H655Y_T1027I_V1176F) P5335S_stab_PP(K986P_V987P_E484K_D614G_V1176F) R10614S_stab_PP(K986P_V987P_S13I_W152C_L452R_D614G) R10520S_stab_PP(K986P_V987P_Q52R_A67V_H69del_V70del_(—)Y144del_E484K_D614G_Q677H_F888L) R10577S_stab_PP(K986P_V987P_A67V_H69del_V70del_Y144del_(—)E484K_D614G_Q677H_F888L) R10598 S_stab_PP(K986P_V987P_L452R_D614G_P681R)P5333 S_stab_PP(K986P_V987P_E154K_L452R_E484Q_D614G_(—) P681R_Q1071H)R10630, S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_(—) R10827**,T478K_D614G_P681R_D950N) R10828**, R11043, R11160**, R11161** R10824,S_stab_PP(K986P_V987P_T19R_F157del_R158del_L452R_(—) R10825**,T478K_D614G_P681R_D950N) R10826** P5336,S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_(—) R10800Y248del_L249del_T250del_P251del_G252del_L452Q_(—) F490S_D614G_T859N)R10575 S_stab_PP(K986P_V987P_H69del_V70del_N439K_D614G) R10579S_stab_PP(K986P_V987P_L5F_T95I_D253G_E484K_D614G_A701V) R10581S_stab_PP(K986P_V987P_L5F_T95I_D253G_S477N_D614G_Q957R) R10592S_stab_PP(K986P_V987P_F157L_V367F_Q613H_P681R) R10615S_stab_PP(K986P_V987P_S254F_D614G_P681R_G769V) P5418S_stab_PP(K986P_V987P_P26S_H69del_V70del_V126A_Y144del_(—)L242del_A243del_L244del_H245Y_S477N_E484K_D614G_P681H_(—) T1027I_D1118H)R10679 S_stab_PP(K986P_V987P_T95I_Y144T_Y145S_ins145N_R346K_(—)E484K_N501Y_D614G_P681H_D950N) P5420S_stab_PP(K986P_V987P_ins214TDR_Q414K_N450K_D614G_T716I) P5416S_stab_PP(K986P_V987P_T478K_D614G_P681H_T732A)S_stab_PP(K986P_V987P_E484K_N501Y_D614G_P681H_E1092K_(—) H1101Y_V1176F)S_stab_PP(K986P_V987P_H66D_G142V_Y144del_Y145del_D215G_(—)V483A_D614G_H655Y_G669S_Q949R_N1187D)S_stab_PP(K986P_V987P_Y144del_L452R_T478K_P681R)S_stab_PP(K986P_V987P_T19R_Y144del_Y145del_L452R_T478K_(—) D614G_P681R)R10922 S_stab_PP(K986P_V987P_P9L_C136F_Y144del_R190S_D215G_(—)L242del_A243del_Y449H_E484K_N501Y_D614G_H655Y_N679K_(—) T716I_T859N)R11175, S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) R11176,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) R11177**,S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—) R11178**N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_(—)N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v1)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_D796Y_(—)N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v1 R11113,S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—) R11114,V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—) R11115**,S371L_S373P_S375F_K417N_N440K_G446S_S477N_T478K_E484A_(—) R11116**Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_(—)P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v0)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_K417N_N440K_G446S_S477N_T478K_E484A_(—)Q493R_G496S_Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_(—)P681H_N764K_D796Y_N856K_Q954H_N969K_L981F);S_stab_PP(K986P_V987P_BA.1_v0)S_stab_PP(K986P_V987P_A67V_T95I_G339D_S371L_S373P_(—)S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_(—)Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_N856K_(—) Q954H_N969K_L981F);S_stab_PP(K986P_V987P_B.1.1.529)S_stab_PP(K986P_V987P_A67V_T95I_G339D_S371L_S373P_(—)S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_N501Y_(—)Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_N856K_(—) Q954H_N969K_L981F);S_stab_PP(K986P_V987P_B.1.1.529)S_stab_PP(K986P_V987P_T19I_L24del_P25del_P26del_(—)A27S_G142D_V213G_G339D_S371F_S373P_S375F_T376A_(—)D405N_S477N_T478K_E484A_Q493R_Q498R_N501Y_Y505H_(—)D614G_H655Y_N679K_P681H_D796Y_Q954H_N969K); S_stab_PP(K986P_V987P_BA.2)S_stab_PP(K986P_V987P_T19I_L24del_P25del_P26del_A27S_(—)G142D_V213G_G339D_S371F_S373P_S375F_T376A_D405N_S477N_(—)T478K_E484A_Q493R_Q498R_N501Y_Y505H_D614G_H655Y_N679K_(—)P681H_D796Y_Q954H_N969K); S_stab_PP(K986P_V987P_BA.2) P5508S_stab_PP(K986P_V987P_G75V_T76I_R246del_S247del_Y248del_(—)L249del_T250del_P251del_G252del_D253N_L452Q_F490S_D614G_T859N) P5453S_stab_PP(K986P_V987P_S12F_H69del_V70del_W152R_R346S_(—)L452R_D614G_Q677H_A899S) P5507S_stab_PP(K986P_V987P_I210T_N440K_E484K_D614G_(—) D936N_S939F_T10271)P5509 S_stab_PP(K986P_V987P_W152L_E484K_D614G_G769V) P5572S_stab_PP(K986P_V987P_T20I_R357K_D614G) P5664S_stab_PP(K986P_V987P_T95I_Y144del_E484K_D614G_P681H_(—) D796H) R10884S_stab_PP(K986P_V987P_T19R_G142D_E156G_F157del_(—)R158del_L452R_T478K_D614G_P681R_D950N) R10801,S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) R10832**,R158del_W258L_K417N_L452R_T478K_D614G_P681R_D950N) R10833** R10829,S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—) R10830**,R158del_W258L_K417N_L452R_T478K_D614G_P681R_D950N) R10831** R10802S_stab_PP(K986P_V987P_T19R_V70F_G142D_E156G_F157del_(—)R158del_A222V_K417N_L452R_T478K_D614G_P681R_D950N) P5662S_stab_PP(K986P_V987P_T19R_T95I_G142D_E156G_F157del_(—)R158del_L452R_T478K_D614G_P681R_D950N) P5663S_stab_PP(K986P_V987P_T19R_E156G_F157del_R158del_(—)L452R_T478K_D614G_P681R_D950N) R11036S_stab_PP(K986P_V987P_T19R_T95I_G142D_Y145H_E156G_(—)F157del_R158del_A222V_L452R_T478K_D614G_P681R_D950N) P5454S_stab_PP(K986P_V987P_T19R_L452R_E484Q_D614G_P681R_D950N)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_N440K_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v2)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_N440K_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v2)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_N856K_(—)Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v3)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_D796Y_N856K_(—)Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v3)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_A701V_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v4)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_S477N_T478K_E484A_Q493R_G496S_Q498R_(—)N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_A701V_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v4)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_G446S_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v5)S_stab_PP(K986P_V987P_A67V_H69del_V70del_T95I_G142D_(—)V143del_Y144del_Y145del_N211del_L212I_ins214EPE_G339D_(—)S371L_S373P_S375F_G446S_S477N_T478K_E484A_Q493R_G496S_(—)Q498R_N501Y_Y505H_T547K_D614G_H655Y_N679K_P681H_N764K_(—)D796Y_N856K_Q954H_N969K_L981F); S_stab_PP(K986P_V987P_BA.1_v5) 5′-UTR/3′-UTR; SEQ ID SEQ ID SEQ ID CDS UTR NO: NO: NO: RNA ID opt. Design3′-end Protein CDS RNA R9488, opt1 HSD17B4/ hSL-A100 1 136  148 R9492,(go) PSMB3; a-1 R9487, opt1 HSD17B4/ hSL-A100 10 137 149, R9491, (go)PSMB3; 28736 R9709, a-1 R10159**, R10160, R10727**, R10820**, R10821**R10166, opt1 HSD17B4/ hSL-A100 22738 22765 22792, R10812**, (go) PSMB3;28737 R10813** a-1 R10811, opt1 HSD17B4/ A100 22738 22765 24838,R10814**, (go) PSMB3; 28827 R10815** a-1 R10279 opt1 HSD17B4/ hSL-A10022740 22767 22794 (go) PSMB3; a-1 R10299 opt1 HSD17B4/ hSL-A100 2274222769 22796 (go) PSMB3; a-1 R10286 opt1 HSD17B4/ hSL-A100 22744 2277122798 (go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 22746 22773 22800 (go)PSMB3; a-1 R10275 opt1 HSD17B4/ hSL-A100 22748 22775 22802 (go) PSMB3;a-1 R10283 opt1 HSD17B4/ hSL-A100 22750 22777 22804 (go) PSMB3; a-1R10291 opt1 HSD17B4/ hSL-A100 22752 22779 22806 (go) PSMB3; a-1 opt1HSD17B4/ hSL-A100 22754 22781 22808 (go) PSMB3; a-1 R10295 opt1 HSD17B4/hSL-A100 22756 22783 22810 (go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 2275822785 22812 (go) PSMB3; a-1 R10162** opt10 HSD17B4/ hSL-A100 10 146  151(go PSMB3; mod) a-1 R10357 opt1 HSD17B4/ hSL-A100 22959 23089 23529 (go)PSMB3; a-1 R10361 opt1 HSD17B4/ hSL-A100 22960 23090 23530 (go) PSMB3;a-1 R10384 opt1 HSD17B4/ hSL-A100 22961 23091 23531 (go) PSMB3; a-1R10385 opt1 HSD17B4/ hSL-A100 22963 23093 23533 (go) PSMB3; a-1 R10410opt1 HSD17B4/ hSL-A100 * * * (go) PSMB3; a-1 R10452 opt1 HSD17B4/ A10022961 23091 * (go) PSMB3; a-1 R9515 opt1 -/muag; A64-N5- 10 137  163(go) i-3 C30-hSL- N5 R10385 opt1 HSD17B4/ hSL-A100 22963 23093 23533(go) PSMB3; a-1 R10616 opt1 HSD17B4/ hSL-A100 27091 27114 27390 (go)PSMB3; a-1 P5335 opt1 HSD17B4/ hSL-A100 27092 27115 27391 (go) PSMB3;a-1 R10614 opt1 HSD17B4/ hSL-A100 22964 23094 23534 (go) PSMB3; a-1R10520 opt1 HSD17B4/ hSL-A100 27089 27112 27388 (go) PSMB3; a-1 R10577opt1 HSD17B4/ hSL-A100 27090 27113 27389 (go) PSMB3; a-1 R10598 opt1HSD17B4/ hSL-A100 27093 27116 27392 (go) PSMB3; a-1 P5333 opt1 HSD17B4/hSL-A100 27094 27117 27393 (go) PSMB3; a-1 R10630, opt1 HSD17B4/hSL-A100 27095 27118 27394, R10827**, (go) PSMB3; 28762 R10828**, a-1R11043, R11160**, R11161** R10824, opt1 HSD17B4/ A100 27095 27118 27532,R10825**, (go) PSMB3; 28852 R10826** a-1 P5336, opt1 HSD17B4/ hSL-A10027096 27119 27395 R10800 (go) PSMB3; a-1 R10575 opt1 HSD17B4/ hSL-A10027097 27120 27396 (go) PSMB3; a-1 R10579 opt1 HSD17B4/ hSL-A100 2709827121 27397 (go) PSMB3; a-1 R10581 opt1 HSD17B4/ hSL-A100 27099 2712227398 (go) PSMB3; a-1 R10592 opt1 HSD17B4/ hSL-A100 27100 27123 27399(go) PSMB3; a-1 R10615 opt1 HSD17B4/ hSL-A100 27101 27124 27400 (go)PSMB3; a-1 P5418 opt1 HSD17B4/ hSL-A100 27102 27125 27401 (go) PSMB3;a-1 R10679 opt1 HSD17B4/ hSL-A100 27103 27126 27402 (go) PSMB3; a-1P5420 opt1 HSD17B4/ hSL-A100 27104 27127 27403 (go) PSMB3; a-1 P5416opt1 HSD17B4/ hSL-A100 27105 27128 27404 (go) PSMB3; a-1 opt1 HSD17B4/hSL-A100 27106 27129 27405 (go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 2710727130 27406 (go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 27108 27131 27407(go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 27109 27132 27408 (go) PSMB3; a-1R10922 opt1 HSD17B4/ hSL-A100 28540 28589 28638 (go) PSMB3; a-1 R11175,opt1 HSD17B4/ hSL-A100 28541 28590 28639, R11176, (go) PSMB3; 28778R11177**, a-1 R11178** opt1 HSD17B4/ A100 28541 28590 28688, (go) PSMB3;28868 a-1 R11113, opt1 HSD17B4/ hSL-A100 28542 28591 28640, R11114, (go)PSMB3; 28779 R11115**, a-1 R11116** opt1 HSD17B4/ A100 28542 2859128689, (go) PSMB3; 28869 a-1 opt1 HSD17B4/ hSL-A100 28543 28592 28641,(go) PSMB3; 28780 a-1 opt1 HSD17B4/ A100 28543 28592 28690, (go) PSMB3;28870 a-1 opt1 HSD17B4/ hSL-A100 28544 28593 28642, (go) PSMB3; 28781a-1 opt1 HSD17B4/ A100 28544 28593 28691, (go) PSMB3; 28871 a-1 P5508opt1 HSD17B4/ hSL-A100 28545 28594 28643 (go) PSMB3; a-1 P5453 opt1HSD17B4/ hSL-A100 28547 28596 28645 (go) PSMB3; a-1 P5507 opt1 HSD17B4/hSL-A100 28548 28597 28646 (go) PSMB3; a-1 P5509 opt1 HSD17B4/ hSL-A10028549 28598 28647 (go) PSMB3; a-1 P5572 opt1 HSD17B4/ hSL-A100 2855028599 28648 (go) PSMB3; a-1 P5664 opt1 HSD17B4/ hSL-A100 28551 2860028649 (go) PSMB3; a-1 R10884 opt1 HSD17B4/ hSL-A100 28552 28601 28650(go) PSMB3; a-1 R10801, opt1 HSD17B4/ hSL-A100 28553 28602 28651,R10832**, (go) PSMB3; 28790 R10833** a-1 R10829, opt1 HSD17B4/ A10028553 28602 28700, R10830**, (go) PSMB3; 28880 R10831** a-1 R10802 opt1HSD17B4/ hSL-A100 28554 28603 28652 (go) PSMB3; a-1 P5662 opt1 HSD17B4/hSL-A100 28555 28604 28653 (go) PSMB3; a-1 P5663 opt1 HSD17B4/ hSL-A10028556 28605 28654 (go) PSMB3; a-1 R11036 opt1 HSD17B4/ hSL-A100 2855728606 28655 (go) PSMB3; a-1 P5454 opt1 HSD17B4/ hSL-A100 28558 2860728656 (go) PSMB3; a-1 opt1 HSD17B4/ hSL-A100 28917 28921 28927, (go)PSMB3; 28935 a-1 opt1 HSD17B4/ A100 28917 28921 28929, (go) PSMB3; 28937a-1 opt1 HSD17B4/ hSL-A100 28918 28922 28926, (go) PSMB3; 28934 a-1 opt1HSD17B4/ A100 28918 28922 28930, (go) PSMB3; 28938 a-1 opt1 HSD17B4/hSL-A100 28919 28923 28927, (go) PSMB3; 28935 a-1 opt1 HSD17B4/ A10028919 28923 28931, (go) PSMB3; 28939 a-1 opt1 HSD17B4/ hSL-A100 2892028924 28928, (go) PSMB3; 28936 a-1 opt1 HSD17B4/ A100 28920 28924 28932,(go) PSMB3; 28940 a-1 **mRNA R10159, R10162, R10157, R10712, R10813,R10815, R10821, R10823, R10826, R10828, R10831, R10833, R11116, R11120,R11161, R11178 were produced with N(1)-methylpseudouridine (m1ψ);R10727, R10728, R10812, R10814, R10820, R10822, R10825, R10827, R10830,R10832, R11115, R11119, R11160, R11177 were produced with pseudouridine(ψ)) Spike prefusion stabilized protein (=S_stab), Spike protein (=S)

1.4. Preparation of an LNP Formulated mRNA Composition:

LNPs were prepared using cationic lipids, structural lipids, aPEG-lipids, and cholesterol. Lipid solution (in ethanol) was mixed withRNA solution (aqueous buffer) using a microfluidic mixing device.Obtained LNPs were re-buffered in a carbohydrate buffer via dialysis,and up-concentrated to a target concentration using ultracentrifugationtubes. LNP-formulated mRNA was stored at −80° C. prior to use in invitro or in vivo experiments.

Lipid nanoparticles were prepared and tested according to the generalprocedures described in PCT Pub. Nos. WO2015/199952, WO2017/004143 andWO2017/075531, the full disclosures of which are incorporated herein byreference. Lipid nanoparticle (LNP)-formulated mRNA was prepared usingan ionizable amino lipid (cationic lipid), phospholipid, cholesterol anda PEGylated lipid. LNPs were prepared as follows. Cationic lipidaccording to formuala III-3 (ALC-0315), DSPC, cholesterol and PEG-lipidaccording to formula IVa (ALC-0159) were solubilized in ethanol at amolar ratio of approximately 47.5:10:40.8:1.7 (see Table 5). Lipidnanoparticles (LNP) comprising compound III-3 were prepared at a ratioof mRNA (sequences see Table 4) to Total Lipid of 0.03-0.04 w/w.Briefly, the mRNA was diluted to 0.05 to 0.2mg/mL in 10 to 50 mM citratebuffer, pH 4. Syringe pumps were used to mix the ethanolic lipidsolution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3(vol/vol) with total flow rates above 15 ml/min. The ethanol was thenremoved and the external buffer replaced with PBS by dialysis. Finally,the lipid nanoparticles were filtered through a 0.2 μm pore sterilefilter. Lipid nanoparticle particle diameter size was 60-90 nm asdetermined by quasi-elastic light scattering using a Malvern ZetasizerNano (Malvern, UK).

TABLE 5 Lipid-based carrier composition of the examples Ratio Compounds(mol %) Structure Mass 1 Cholesterol 40.9

386.4 2 1,2-distearoyl- sn-glycero-3- phosphocholine (DSPC) 10

789.6 3 Cationic Lipid 47.4

765.7 4 PEG Lipid 1.7

2010.1 Average n = ~49

1.5. Preparation of Combination mRNA Vaccines Comprising AntigenCombinations (Bivalent or Multivalent Vaccine Compositions):

Combination mRNA vaccines were formulated with LNPs either in a separateor co-formulated way. For separately mixed or formulated mRNA vaccines,each mRNA component was prepared and separately LNP formulated asdescribed in Example 1.4, followed by mixing of the differentLNP-formulated components. For co-formulated mRNA vaccine, the differentmRNA components are firstly mixed together, followed by a co-formulationin LNPs as described in Example 1.4.

Example 2 Multivalency Study in Rats: Immunogenicity of a BivalentCV2CoV (R9709) and CV2CoV.351 (R10384) Vaccine upon i.m. Administrationin Wistar Rats

In this study, the humoral immunogenicity induced by LNP-formulatedbivalent mRNA vaccine CV2CoV/CV2CoV.351 (R9709/R10384) was evaluated inWistar rats in comparison to CV2CoV (R9709) or CV2CoV.351 (R10384).

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized ancestral SARS-CoV-2 Sand CV2CoV.351-mRNA based SARS-CoV-2 vaccine encoding for full length,pre-fusion stabilized SARS-CoV-2 B.1.351 S) were prepared as describedin Example 1.2 (RNA in vitro transcription). HPLC purified mRNA wasformulated with LNPs according to Example 1.4 and Example 1.5(separately mixed or formulated “mixed 2 LNPs” or co-formulated “mixed 1LNP” for bivalent mRNA vaccines (group F, G, H) prior to use in in vivovaccination experiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 6. Buffer vaccinated animals served as anegative control (group A). All animals were vaccinated on day 0 and day21. Blood samples were collected on day 14, day 21 and day 42 for thedetermination of humoral immune responses.

TABLE 6 Vaccination regimen (Example 2) 1st 2nd Immunisation Serum GroupAnimals vaccination vaccination schedule isolation A N = 4 NaCl NaCl BWistar CV2CoV 8 μg CV2CoV 8 μg D 0, D 14 C Female CV2CoV.351 8 μgCV2CoV.351 8 μg D 21 D 21 D N = 6 CV2CoV 8 μg CV2CoV.351 8 μg D 42 ECV2CoV.351 8 μg CV2CoV 8 μg F CV2CoV + CV2CoV + CV2CoV.351 CV2CoV.351mixed 1 LNP 8 μg mixed 1 LNP 8 μg G CV2CoV + CV2CoV + CV2CoV.351CV2CoV.351: mixed 2 LNPs 8 μg mixed 2 LNPs 8 μg H CV2CoV CV2CoV (leftleg) 4 μg (left leg) 4 μg CV2CoV.351 CV2CoV.351 (right leg) 4 μg (rightleg) 4 μg CV2CoV is shown as R9709 in Table 4 and CV2CoV.351 is shown asR10384 in Table 4

Determination of IgG1 and IgG2 Spike-Binding Antibody Titers UsingELISA:

Anti-SARS-CoV-2 Spike RBD protein specific binding antibodies, displayedas endpoint titers for IgG1 and IgG2a, were determined in sera isolatedon day 14 and day 21. Recombinant SARS-CoV-2 Spike RBD protein orrecombinant SARS-CoV-2 B.1.351 Spike protein RBD (K417N, E484K, N501Y)was used for coating. Coated plates were incubated using respectiveserum dilutions, and binding of specific antibodies to RBD were detectedwith a biotinylated antibody.

Determination of VNTs

For the analysis of VNTs of rat sera, serial dilutions ofheat-inactivated sera (56° C. for 30 min) tested in duplicates with astarting dilution of 1:10 followed by 1:2 serial dilutions wereincubated with 100 TCID50 of SARS-CoV-2. For this, different viruseswere employed:

-   -   ancestral SARS-CoV-2: strain 2019-nCov/Italy-INMI derived from        the EVAg    -   B.1.351 variant SARS-CoV-2: strain        hCoV-19/Netherlands/NoordHolland_10159/2021, South African        variant, nextstrain Glade 20H, lineage B.1.351 supplied by the        EVAg    -   B.1.1.7 variant SARS-CoV-2: strain 14484 human swab isolated by        VisMederi Research, which contains the following mutations        compared to ancestral virus: N501Y, A570D, T572I, D614G, P681H,        T716I, S735L, S982A, D1118H. Of note, these mutations differ        from the consensus sequence of the variant4, i.e. deletions        dH69/V70 and dY144 are missing and T572I and S735L represent        additional mutations.    -   P.1 variant SARS-CoV-2 strain: PG_253 isolated by University of        Siena, containing the following mutations: L18F T20N P26S D138Y        R190S K417T E484K N501Y D614G H655Y T1027I and V1176F    -   of wild type SARS-CoV-2 (strain 2019-nCov/Italy-INMI derived        from the EVAg) or the B.1.351 variant SARS-CoV-2 (strain        hCoV-19/Netherlands/NoordHolland_10159/2021, South African        variant, next strain clade 20H, lineage B.1.351 supplied by the        EVAg)

Virus was incubated for 1 hour at 37° C. Every plate contained adedicated row (8 wells) for cell control, which contains only cells andmedium, and a dedicated row of virus control, which contains only cellsand virus. Infectious virus was quantified upon incubation of 100 μl ofvirus-serum mixture with a confluent layer of Vero E6 cells (ATCC,Cat.1586) followed by incubation for 3 days (ancestral SARS-CoV-2) or 4days (SARS-CoV-2 B.1.351, B.1.1.7 and P.1) at 37° C. and microscopicalscoring for CPE formation. A back titration was performed for each runin order to verify the correct range of TCID50 of the working virussolution. VN titers were calculated according to the method described byReed & Muench. If no neutralization was observed (MNt<10), an arbitraryvalue of 5 was reported. Analyses were carried out at VisMederi srl(Siena, Italy).

Results:

As shown in FIG. 1 significant IgG1 and IgG2a binding antibody responsesto the receptor binding domain (RBD) of ancestral SARS-CoV-2 and the RBDof the B.351 variant on day 14 (FIG. 1A-D) and on day 21 (FIG. 1E-H)were detected for the group vaccinated with the CV2CoV andCV2CoV.351. Onday 14, as shown in FIG. 1A, comparable IgG1 response for all groups(ancestral receptor binding domain (RBD) protein coating) and comparableIgG2a titers (FIG. 1B) for all groups were detected (ancestralSARS-CoV-2 receptor binding domain (RBD) protein coating). On day 14,shown in FIG. 1C, comparable IgG1 response for all vaccination designs(RBD B.1.351 variant K417N, E484K, N501Y protein coating) and comparableIgG2a titers (FIG. 1D) for all vaccination designs were detected (RBDB.1.351 variant K417N, E484K, N501Y protein coating). On day 21, asshown in FIG. 1E, comparable IgG1 response for all vaccination designs(ancestral SARS-CoV-2 receptor binding domain (RBD) protein coating) andcomparable IgG2a titers (FIG. 1F) for all vaccination designs weredetected (ancestral SARS-CoV-2 receptor binding domain (RBD) proteincoating). On day 21, as shown in FIG. 1G, comparable IgG1 response forall vaccination designs (RBD B.1.351 variant K417N, E484K, N501Y proteincoating) and comparable IgG2a titers (FIG. 1H) for all vaccinationdesigns were shown (RBD B.1.351 variant K417N, E484K, N501Y proteincoating). Overall, CV2CoV and CV2CoV.351 given alone (group B-C), insequential combination or (group D-E) and as co-deliveries of bothvaccine variants (bivalent vaccine CV2CoV/CV2CoV.351) in one and twoLNPs, injected in the same leg or different legs (group F-H) inducedcomparable levels of binding antibodies to the ancestral SARS-CoV-2 RBDand the RBD of the B.1.351 variant.

As shown in FIG. 2A (day 14) and 2B (day 21) the vaccination with CV2CoVand CV2CoV.351 induced significant levels of VNTs against ancestralSARS-CoV-2 in rats (group B-H). Overall, vaccination with either vaccinealone or in combination (group B-H) generated high VNT levels.Especially two vaccinations with CV2CoV in in group B, sequentialvaccination with CV2CoV and CV2CoV.351 in group D and co-delivery ofboth vaccine variants (bivalent vaccine CV2CoV/CV2CoV.351) intodifferent legs (group H) showed increased VNTs at these early timepoints. On day 42, increased levels of VNT were detected for all groups(group B-H) (FIG. 2C). The bivalent vaccines (group F-H) inducedresponses that were comparable to the monovalent vaccines (groups B-E)despite using lower doses of each vaccine on day 42.

As shown in FIG. 3A (day 14) and 3B (day 21) the vaccination with CV2CoVand CV2CoV.351 induced significant levels of VNTs against B.1.351variant SARS-CoV-2 (group B-H). Overall, vaccination with either vaccinealone or in combination (group B-H) generated sufficient VNT levels. Twovaccinations with CV2CoV.351 in group C, sequential vaccination withCV2CoV.351 and CV2CoV in group E and co-delivery of both vaccinevariants (bivalent vaccine CV2CoV/CV2CoV.351) into different legs (groupH) showed increased VNTs at early time points. On day 42, increasedlevels of VNTs were shown for all groups (group B-H) (FIG. 3C). Thebivalent vaccines (group F-H) induced responses that were comparable tothe monovalent vaccines (groups B-E) despite using lower doses of eachvaccine on day 42.

As shown in FIG. 4 significant induction of VNTs assessed in a CPE-basedassay for all groups (group B-H) using B.1.1.7 variant SARS-CoV-2 (FIG.4A) or B.1.1.28 P.1 (FIG. 4B) were detected on day 42. Co-delivery ofboth vaccine variants into the same leg (group F and G) or intodifferent legs (group H) can generate responses against both variants onday 42. Table 7 summarizes median VNTs against ancestral, B.1.1.7,B.1.351 and P1 SARS-CoV-2 variants on day 42.

TABLE 7 Median VNTs (day 42) VNTs VNTs VNTs VNTs Group ancestral B.1.1.7B.1.351 P.1 A 5 5 5 5 B 3620 4370 2185 4370 C 1065 1225 13860 9801 D1970 2263 4526 4526 E 1545 3090 8740 8740 F 2560 2560 9801 8740 G 43705120 12361 14481 H 4370 6180 12361 14481

Overall, the bivalent vaccine elicited robust levels of both RBD bindingand virus neutralizing antibodies that were able to neutralize bothancestral and B.1.351 SARS-CoV-2 as well as the variants B.1.1.7 andB.1.1.28 P.1.

Example 3 Dose Response Study: Immunogenicity of CV2CoV.351 inComparison to CV2CoV upon i.m. Administration in Wistar Rats

The objective of this study was to assess immunogenicity and earlyinnate stimulation of the CV2CoV.351 vaccine in rats in a dose-responsestudy.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-LNP formulated mRNA based SARS-CoV-2vaccine encoding for full length, pre-fusion stabilized ancestralSARS-CoV-2 S and CV2CoV.351-LNP formulated mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized SARS-CoV-2 B.1.351 S)were prepared as described in Example 1.2 (RNA in vitro transcription).HPLC purified mRNA was formulated with LNPs according to Example 1.4prior to use in in vivo vaccination experiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 8. Buffer vaccinated animals served as anegative control (group A). All animals were vaccinated on day 0 and day21. Blood samples were collected day 14, day 21 and day 42 for thedetermination of antibody titers.

TABLE 8 Vaccination regimen (Example 3) i.m. mRNA vacci- Blood GroupAnimals Vaccine Dose nation collection A Wistar Neg. Ctrl / D 0, D 14,Female (Buffer) D 21 D 21, N = 4 D 42 B Wistar CV2CoV.351 0.5 μg CFemale (B.1.351 2 μg D N = 6 Variant S) 8 μg E 40 μg F CV2CoV 0.5 μg G(ancestral S) 2 μg H 8 μg I 40 μg CV2CoV is shown as R9709 in Table 4and CV2CoV.351 is shown as R10384 in Table 4

Determination of IgG1 and IgG2 spike-binding antibody titers using ELISAand determination of VNTs were performed as described in Example 2.

Results:

Antigen-specific binding antibody titers (analyzed via ELISA) and VNTsagainst both ancestral and B.1.351 SARS-CoV-2 were detectable in a dosedependent manner in animals vaccinated with both vaccines. Binding aswell as neutralizing antibodies increased over time and with increasingthe dose.

As shown in FIG. 5A (IgG1) and 5B (IgG2a) (ancestral SARS-CoV-2 RBDcoating) and 5C (IgG1) and 5D (IgG2a) (B.1.351 variant RBD K417N, E484K,N501Y coating) vaccination with CV2CoV (group B-E) and CV2CoV.351 (groupF-I) induced spike-binding antibody titers using doses of 0.5 μg, 2 μgn8 μg and 40 μg in rats on day 14. As shown in FIG. 5E (IgG1), FIG. 5F(IgG2a) (ancestral SARS-CoV-2 RBD coating) and FIG. 5G (IgG1) and 5H(IgG2a) (B.1.351 variant RBD K417N, E484K, N501Y coating) vaccinationwith CV2CoV (group B-E) and CV2CoV.351 (group F-I) induced spike-bindingantibody titers in rats using doses of 0.5 μg, 2 μg and 8 μg and 40 μgon day 21.

As shown in FIG. 6A the B.1.351 variant vaccine CV2CoV.351 (group B-E)induced dose-dependent VNTs against ancestral SARS-CoV-2 (heterologousresponse) on day 14 in all dose groups. Compared to responses uponvaccination with CV2CoV (homologous response), VNTs in CV2CoV.351vaccinated groups are decreased by a factor of approx. 2 on day 14. FIG.6B shows that CV2CoV.351 (group B-E) induces dose-dependent VNTs againstB.1.351 SARS-CoV-2 (homologous response) on day 14 in all dose groups.CV2CoV.351 vaccination elicited high levels of VNTs against homologousvirus that were 45× increased on day 14, compared to heterologous VNTsagainst ancestral virus (average difference of all dose groups). Incomparison to vaccination with CV2CoV (group F-I), VNTs induced byCV2CoV.351 were increased by a factor of 41 on day 14 (averagedifference of all dose groups). As shown in FIG. 6C the B.1.351 variantvaccine CV2CoV.351 (group B-E) induced dose-dependent VNTs againstancestral SARS-CoV-2 (heterologous response) on day 21 in all dosegroups. Compared to responses upon vaccination with CV2CoV (homologousresponse), VNTs in CV2CoV.351 vaccinated groups are decreased by afactor of approx. 2 on day 21. As shown in FIG. 6D CV2CoV.351 inducesslightly dose-dependent VNTs against B.1.351 SARS-CoV-2 (homologousresponse) on day 21 in all dose groups. CV2CoV.351 vaccination elicitedhigh levels of VNTs against homologous virus that were 35× increased onday 21, compared to heterologous VNTs against ancestral virus (averagedifference of all dose groups). In comparison to vaccination withCV2CoV, VNTs induced by CV2CoV.351 were increased by a factor of 42 onday 21 (average difference of all dose groups). As shown in FIG. 6E theB.1.351 variant vaccine CV2CoV.351 induced VNTs against ancestralSARS-CoV-2 (heterologous response) on day 41 in all dose groups.Slightly higher responses except for 0.5 μg dose group (group F) wereshown upon vaccination with CV2CoV (homologous response). As shown inFIG. 6F CV2CoV.351 induced VNTs against B.1.351 SARS-CoV-2 (homologousresponse) on day 42 in all dose groups. In comparison to vaccinationwith CV2CoV, VNTs induced by CV2CoV.351 were increased on day 42. Asshown in FIG. 6G the B.1.351 variant vaccine CV2CoV.351 induced VNTsagainst B. 1.1.7 variant SARS-CoV-2 (heterologous response) on day 42 inall dose groups. Similar responses were shown upon vaccination withCV2CoV (heterologous response). As shown in FIG. 6H CV2CoV.351 inducedVNTs against or B.1.1.28 P.1 SARS-CoV-2 (homologous response) on day 42in all dose groups. Lower responses were detected upon vaccination withCV2CoV (heterologous response).

Overall, the SARS-CoV-2 B.1.351 variant mRNA vaccine candidateCV2CoV.351 induced robust humoral immune responses in rats, asdetermined by binding and virus neutralizing antibody titers. Virusneutralizing titers against B.1.351 were substantially increased uponvaccination with CV2CoV.351 compared to vaccination with CV2CoV.

Example 4 Extended Multivalency Vaccination Study: Immunogenicity of aBivalent CV2CoV and CV2CoV.351 Vaccine upon i.m. Administration inWistar Rats (Prophetic)

The objective of this study is to assess immunogenicity and early innatestimulation of the bivalent CV2CoV/CV2CoV.351 vaccine in a thirdvaccination in rats.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-LNP formulated mRNA based SARS-CoV-2vaccine encoding for full length, pre-fusion stabilized ancestralSARS-CoV-2 S and CV2CoV.351-LNP formulated mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized SARS-CoV-2 B.1.351 S)are prepared as described in Example 1.2 (RNA in vitro transcription).HPLC purified mRNA is formulated with LNPs according to Example 1.4 andExample 1.5 (separately mixed or formulated for bivalent mRNA vaccines)prior to use in in vivo vaccination experiments.

Immunization:

Rats are injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 9. Buffer vaccinated animals serve as anegative control (group A). All animals are vaccinated on week 0, week 3(day 21) and for group B additionally on week 15 (day 105). Bloodsamples are collected on day 0, day 14, day 21, day 42, day 77, day 105,day 119 and day 133 for the determination of antibody titers.

TABLE 9 Vaccination regimen (Example 4) 1st 2nd 3rd vaccinationvaccination vaccination Serum Group Animals week 0 week 3 week 15isolation A N = 4 NaCl NaCl NaCl D 0 (18 h), B Wistar CV2CoV CV2CoVCV2CoV + Week 2 (D 14) Female 8 μg 8 μg CV2CoV.351 8 μg Week 3 (D 21) N= 6 (mixed 2 LNPs) Week 6 (D 42) C CV2CoV.351 CV2CoV.351 Week 11 (D 77)8 μg 8 μg Week 15 (D 105) D CV2CoV CV2CoV.351 Week 17 (D 119) 8 μg 8 μgWeek 19 (D 133) E CV2CoV.351 CV2CoV.351 8 μg 8 μg F CV2CoV+ CV2CoV +CV2CoV.351 CV2CoV.351 mixed 1 LNP mixed 1 LNP 8 μg 8 μg G CV2CoV +CV2CoV+ CV2CoV.351 CV2CoV.351: mixed 2 LNPs mixed 2 LNPs 8 μg 8 μg HCV2CoV (left leg) CV2CoV (left leg) 4 μg 4 μg CV2CoV.351 CV2CoV.351(right leg) (right leg) 4 μg 4 μg CV2CoV is shown as R9709 in Table 4and CV2CoV.351 is shown as R10384 in Table 4

Determination of IgG1 and IgG2 spike-binding antibody titers using ELISAand determination of VNTs are performed as described in Example 2.

Example 5 Co-Delivery of Vaccines: Vaccination of Rats with mRNAEncoding Ancestral SARS-CoV2 Antigen (CV2CoV) and SARS-CoV2 Antigen ofVariant B.1.351 (CV2CoV.351)

Within this study, the antibody response against both ancestral andB.1.351 SARS-CoV-2 were measured.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized ancestral SARS-CoV-2 Sand CV2CoV.351-mRNA based SARS-CoV-2 vaccine encoding for full length,pre-fusion stabilized SARS-CoV-2 B.1.351 S) were prepared as describedin Example 1.2 (RNA in vitro transcription). HPLC purified mRNA wasformulated with LNPs according to Example 1.4 and Example 1.5(separately mixed or formulated for bivalent mRNA vaccines) prior to usein in vivo.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 10. As a negative control, one group ofrats was vaccinated with buffer (group A). All animals were vaccinatedon week 0 and week 3 (day 21). Blood samples were collected on day 0,day 14, day 21, and day 42 for the determination of antibody titers.

TABLE 10 Vaccination regimen (Example 5) 1st 2nd vaccination vaccinationImmunisation Serum Group Animals week 0 week 3 schedule isolation AWistar NaCl NaCl Week 0 D 0 (18 h), Female Week 3 Week 2 (D 14) N = 4Week 3 (D 21) B Wistar CV2CoV + CV2CoV + Week 6 (D 42) Female CV2CoV.351CV2CoV.351 N = 6 8 μg 8 μg C CV2CoV + CV2CoV + CV2CoV.351 CV2CoV.351 2μg 2 μg D CV2CoV + CV2CoV + CV2CoV.351 CV2CoV.351 0.5 μg 0.5 μg CV2CoVis shown as R9709, CV2CoV.351 as R10384 in Table 4

Determination of IgG1 and IgG2a spike-binding antibody titers usingELISA and determination of VNTs were performed as described in Example2.

Results:

Overall, the bivalent vaccine composition CV2CoV+CV2CoV.351 inducedcomparable levels of spike-binding antibodies to ancestral and B.1.351variant RBD on day 14 (FIG. 7A-7D). Significant levels of spike-bindingantibodies were detectable in all animals vaccinated with 2 μg or 8 μgof bivalent vaccine composition CV2CoV+CV2CoV.351 on day 14 postinjection. Dose dependent levels of IgG1 and IgG2a spike-bindingantibody titers were induced in all groups injected with 0.5 μg, 2 μg or8 μg of bivalent vaccine composition CV2CoV+CV2CoV.351. The ratiosbetween IgG1 and IgG2a antibodies showed a slightly lower induction ofIgG2a antibodies compared to IgG1 for the 2 μg dose. FIGS. 7A and 7Bshows binding antibodies to ancestral SARS-CoV-2 RBD and FIGS. 7C and 7Dto B.1.351 variant RBD. Robust VNTs were induced against ancestralSARS-CoV-2 in a dose dependent manner for the 2 and 8 μg groups overtime (FIG. 7E (day 14), 7F (day 21), and 7I (day 42)) and againstB.1.351 variant SARS-CoV-2 (FIG. 7G (day 14), 7H (day 21), and 7J (day42)). FIGS. 7K and 7L shows the dose dependent induction of VNTs againstvariant B.1.1.7 and P.1 respectively, for the 2 μg and 8 μg dose groups.

Example 6 Booster Study: Vaccination of Rats with Bivalent CVnCoV andCVnCoV.351 Vaccine upon i.m. Administration in Wistar Rats

This study was designed to determine if a homologous boost is able toelicit significant increases of immune response against a heterologousvariant (B.1.351).

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-LNP formulated mRNA based SARS-CoV-2vaccine encoding for full length, pre-fusion stabilized ancestralSARS-CoV-2 S and CV2CoV.351-LNP formulated mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized SARS-CoV-2 B.1.351 S)were prepared as described in Example 1.2 (RNA in vitro transcription).As indicated in Tables 4 and 11, in some constructs, uridine wasreplaced by 1-Methylpseudouridinie. HPLC purified mRNA was formulatedwith LNPs according to Example 1.4 prior to use in in vivo.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 11. As a negative control, one group ofrats was vaccinated with buffer (group A). All animals were vaccinatedin week 0 (day1) and week 3 (day 21) with the same vaccine composition.In week 15 the animal received a third vaccination partially with adifferent vaccine composition. Blood samples were collected on day 0,day 15, day 21, day 42, day 77, day 105, day 119, and day 133 for thedetermination of VNTs.

TABLE 11 Vaccination regimen (Example 6) 1st 2nd 3rd vaccinationvaccination vaccination Immunisation Serum Group Animals week 0 week 3week 15 Dose schedule isolation A N = 4 NaCl NaCl NaCl / D 0, D 0, BWistar m1ψ m1ψ CV2CoV 8 μg D 21, D 15 Female ancestral S ancestral Sancestral S D 105 (3 M) D 21 N = 6 (R10162) (R10162) D 42 C m1ψ m1ψCV2CoV 351 D 119 ancestral S ancestral S Variant S D 105 (R10162)(R10162) D 133 D CV2CoV CV2CoV CV2CoV ancestral S ancestral S ancestralS E CV2CoV CV2CoV CV2CoV.351 ancestral S ancestral S Variant S F CVnCoVCVnCoV CV2CoV ancestral S ancestral S ancestral S G CVnCoV CVnCoVCV2CoV.351 ancestral S ancestral S Variant S CV2CoV is shown as R9709 inTable 4 and CV2CoV.351 is shown as R10384 Table 4

Determination of VNTs were performed as described in Example 2.

Results:

Overall, boosting with either CV2CoV or CV2CoV.351 three months post twoprime vaccinations with CVnCoV, CV2CoV or “m1ψ ancestral S” (R10162)induced a significant increase of virus neutralizing antibodies againstboth ancestral and B.1.351 SARS-CoV-2 (FIGS. 8A and 8B, respectively).Boosting with both CV2CoV and CV2CoV.351 induced VNTs that were able toneutralize ancestral SARS-CoV-2 and SARS-CoV-2 B.1.351, B.1.1.7 and P.1variants (FIG. 8C-8F).

VNTs Against Ancestral SARS-CoV-2 (FIG. 8A):

CVnCoV induced robust VNTs against ancestral SARS-CoV-2 upon twovaccinations in rats (groups G and F). Titers remained readilydetectable until boosting on d105 with a small decrease of titersmeasured over time. CV2CoV (homologous vaccine) showed high boostingcapacity on the background of CVnCoV prime vaccination (group F): VNTsagainst ancestral SARS-CoV-2 were significantly increased by a factor of109 (d105 vs d119) upon CV2CoV boosting.

Compared to responses upon boosting with CV2CoV, titers induced byboosting with CV2CoV.351 (heterologous vaccine) against ancestralSARS-CoV-2 were lower (group G). The observed difference between titersdetected on d105 and d119 amounted to an increase of 6 fold. However,the difference was not statistically significant. Similar results can beachieved with prime vaccinations with CV2CoV or “m1ψ ancestral S”(R10162), whereby the titers before the third (“boosting) vaccination onday 105 are significantly increased compared to CVnCoV induced VNTs.

VNTs Against SARS-CoV-2 B.1.351 (FIG. 8B):

CVnCoV induced robust VNTs against SARS-CoV-2 B.1.351 upon twovaccinations in rats. However, titers were overall lower than againstancestral SARS-CoV-2 (comparing FIG. 8B with FIG. 8A). Boosting withCV2CoV.351 (homologous vaccine) induced a significant increase of 109fold compared to VNTs detected on d105 vs d119 (group G).

CV2CoV (heterologous vaccine) showed a high boosting capacity on thebackground of CVnCoV prime vaccination (group F): VNTs againstSARS-CoV-2 B.1.351 were significantly increased by a factor of 256 (d105vs d119) upon CV2CoV boosting.

Similar results can be achieved with prime vaccinations with CV2CoV or“m1ψ ancestral S” (R10162), whereby the titers before the third(“boosting) vaccination are significantly increased compared to CVnCoVinduced VNTs.

Virus-neutralizing responses against ancestral SARS-CoV-2 as well asagainst SARS-CoV-2 B.1.1.7 (alpha), B.1.351 (beta) and P.1 (gamma)variants were tested 14 days after boosting (FIG. 8C-8F).

Boosting with CV2CoV.351 (homologous vaccine, groups C, E, and G)induced not only strong VNTs against SARS-CoV-2 B.1.351 on d119 (FIG.8I)), but also against the ancestral SARS-CoV-2 (FIG. 8C), SARS-CoV-2B.1.1.7 (FIG. 8E) and P.1 (FIG. 8F) (heterologous vaccine).

Example 7 Vaccination of Mice with mRNA Vaccines Encoding SARS-CoV-2Variants

This study was designed to determine if vaccinations with mRNA vaccinesencoding SARS-CoV-2 variants induce immunogenicity withcross-neutralizing capacity.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs CV2CoV-mRNA based SARS-CoV-2 vaccine encodingfor full length, pre-fusion stabilized ancestral or variant SARS-CoV-2were prepared as described in Example 1.2 (RNA in vitro transcription).HPLC purified mRNA was formulated with LNPs according to Example 1.4prior to use in in vivo.

Immunization:

Mice were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 12. As a negative control, one group ofmice was vaccinated with buffer (group 1). All animals were vaccinatedon day 0 and day 21. Blood samples were collected on day 0, day 14, day21, and day 42 for the determination of antibody titers.

TABLE 12 Vaccination regimen (Example 7) Immunisation Serum GroupAnimals mRNA Dose schedule isolation 1 N = 8 Neg. control: NaCl / D 0, D0 2 Balb/c R9709 CV2CoV (ancestral) 1 μg D 21 D 14 3 female R10357B1.1.7 (alpha) D 21 4 R10384 B1.351 (beta) CV2CoV.351 D 42 5 R10410B.1.1.7 + E484K 6 R10385 P.1 (gamma) 7 R10452 B.1.351 (beta)

Determination of IgG1 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S protein (ancestralSARS-CoV-2 RBD or B.1.351 RBD variant (K427N, E484K, N501Y)) forcoating. Coated plates were incubated using respective serum dilutions,and binding of specific antibodies to SARS-CoV-2 RBD or RBD variant weredetected using biotinylated isotype specific anti-mouse antibodiesfollowed by streptavidin-HRP (horse radish peroxidase) with Amplex assubstrate. Endpoint titers of antibodies were measured by ELISA on day14 post prime vaccination.

Determination of VNTs:

Determination of VNTs assessed in a CPE-based assay was performed asdescribed in Example 2. For detection of VNTs against the SARS-CoV-2delta variant, the virus strainhCoV-19/France/IDF-APHP-HEGP-20-23-2131905084/2021|EPI_ISL_2029113|2021-04-27was used comprising the following mutations: T19R E156G d157F d158RL452R T478K D614G P681R D950N.

Intracellular Cytokine Staining:

Splenocytes from vaccinated mice were isolated on day 42 according to astandard protocol known in the art. Briefly, isolated spleens weregrinded through a cell strainer and washed in PBS/1% FBS followed by redblood cell lysis. After an extensive washing step with PBS/1% FBS,splenocytes were seeded into 96-well plates (2×106 cells per well).Cells were stimulated with a mixture of SARS-CoV-2 ancestral S proteinspecific peptides (1 μg/ml) in the presence of 2.5 μg/ml of an anti-CD28antibody (BD Biosciences) for 6 hours at 37° C. in the presence of aprotein transport inhibitor. The same procedure was repeated forstimulating the splenocytes with a mixture of SARS-CoV-2 B.1.351 Sprotein specific peptides. After stimulation, cells were washed andstained for intracellular cytokines using the Cytofix/Cytoperm reagent(BD Biosciences) according to the manufacturer's instructions. Thefollowing antibodies were used for staining: Thy1.2-FITC (1:200),CD8-APC-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience),CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated withFcγ-block diluted 1:100. Aqua Dye was used to distinguish live/deadcells (Invitrogen). Cells were acquired using a ZE5 flow cytometer(Bio-Rad). Flow cytometry data was analyzed using FlowJo softwarepackage (Tree Star, Inc.).

Results:

On day 14, all tested SARS-CoV-2 mRNA vaccine constructs encoding forfull length, pre-fusion stabilized ancestral or variant SARS-CoV-2induced high IgG antibody responses to ancestral SARS-CoV-2 RBD (FIG.9A) and the B.1.351 variant RBD (K417N, E484K, N501Y) FIG. 9B.

All of the tested mRNA vaccine constructs showed a strong induction ofVNTs on day 42, which is most prominent and also detectable on earliertime points (d14, d21) for homologous neutralization (FIG. 9C: group 2,ancestral; FIG. 9D: group 3, B.1.1.7; FIG. 9E: group 4 and 7, B.1.351;FIG. F: group 6, P1).

As shown in FIG. 9G-J the vaccination with mRNA encoding the differentvariant full length S stabilized proteins induced robust levels ofantigen-specific CD4⁺ and CD8⁺ IFN□/TNF double positive T cells aftertwo vaccinations on day 42 to a similar extent upon stimulation ofsplenocytes with ancestral (FIGS. G and H) or B.1.351 (FIGS. I and J)peptide libraries.

Example 8 Vaccination of Mice with mRNA Vaccines Encoding SARS-CoV-2Variants

This study was designed to determine if vaccination with mRNA vaccinesencoding SARS-CoV-2 variants induces immunogenicity withcross-neutralizing capacity.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-LNP formulated mRNA based SARS-CoV-2vaccine encoding for full length, pre-fusion stabilized ancestral orvariant SARS-CoV-2 S (S_stab) were prepared as described in Example 1.2(RNA in vitro transcription). HPLC purified mRNA was formulated withLNPs according to Example 1.4 prior to use in in vivo.

Immunization:

Mice were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 13. As a negative control, one group ofmice was vaccinated with buffer (group 13). All animals were vaccinatedon day 0 and day 21. Blood samples were collected on day 0, day 14, day21, and day 42 for the determination of antibody titers.

TABLE 13 Vaccination regimen (Example 8) Immunisation Serum GroupAnimals mRNA Dose schedule isolation 1 N = 8 R9709 1 μg D 0, D 0 2Balb/c R10384 B.1.351 (Beta, South Africa) D 21 D 14 3 female R10631E484K, D614G D 21 4 R10614 B.1.429 (Epsilon, USA) D 42 5 R10520 B.1.525(Eta, multiple) 6 R10577 B.1.525_v2 (Eta, multiple) 7 R10575 B.1.258(Czech Republic) 8 R10579 B.1.526_v1 (lota, USA) 9 R10581 B.1.526_v2(lota, USA) 10 R10592A.23.1_v1 (Rwanda/Uganda) 11 R10616P.1_v2 (Gamma,Brazil) 12 R10360 B.1.617_v2 8 (Delta, India) 13 Neg. control: NaCl —

Determination of VNTs with homologous and heterologous variants can beperformed as described in Example 2. For T-cell analysis splenocyteswere isolated on day 42.

T-Cell Analysis by Intracellular Cytokine Staining (ICS):

Splenocytes from vaccinated mice were isolated according to a standardprotocol known in the art. Briefly, isolated spleens were grindedthrough a cell strainer and washed in PBS/1% FBS followed by red bloodcell lysis. After an extensive washing step with PBS/1% FBS, splenocyteswere seeded into 96-well plates (2×10⁶ cells per well). Cells werestimulated with a mixture of SARS-CoV-2 ancestral S protein specificpeptides (1 μg/ml) in the presence of 2.5 μg/ml of an anti-CD28 antibody(BD Biosciences) for 6 hours at 37° C. in the presence of a proteintransport inhibitor. After stimulation, cells were washed and stainedfor intracellular cytokines using the Cytofix/Cytoperm reagent (BDBiosciences) according to the manufacturer's instructions. The followingantibodies were used for staining: Thy1.2-FITC (1:200), CD8-APC-Cy7(1:200), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD HorizonV450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted1:100. Aqua Dye was used to distinguish live/dead cells (Invitrogen).Cells were acquired using a ZE5 flow cytometer (Bio-Rad). Flow cytometrydata was analyzed using FlowJo software package (Tree Star, Inc.).

Results:

As shown in FIG. 10 the vaccination with mRNA encoding the differentvariant full length S stabilized proteins induced robust levels ofantigen-specific CD4⁺ and CD8⁺ IFNγ/TNF double positive T cells (FIGS.10A and B respectively) after two vaccinations on day 42 uponstimulation of splenocytes with ancestral peptide library. It seemslikely that the humoral responses (ELISA or VNTs) would behave in asimilar way as shown in Example 7 (not yet tested for the vaccineconstructs of Table 13).

Example 9A Vaccination of Mice with mRNA Vaccines Encoding SARS-CoV-2Variants (Prophetic)

Within this study, it can be determined if vaccination with mRNAvaccines encoding SARS-CoV-2 variants induces immunogenicity withcross-neutralizing capacity.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized ancestral or variantSARS-CoV-2 S (S_stab) are prepared as described in Example 1.2 (RNA invitro transcription). HPLC purified mRNA is formulated with LNPsaccording to Example 1.4 prior to use in in vivo.

Immunization:

Mice are injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 14A. As a negative control, one group ofmice is vaccinated with buffer (group 1). All animals are vaccinated onday 0 and day 21. Blood samples are collected on day 0, day 14, day 21,and day 42 for the determination of antibody titers.

TABLE 14A Vaccination regimen Immunisation Serum Group Animals mRNA Doseschedule isolation 1 N = 8 Neg. control: 0.9% NaCl — 2 Balb/c R9709(ancestral) 1 μg D 0 D 0 3 female R10800 C37.1 (Lambda, Peru) D 21 D 144 R10679 B.1.621 (Mu, Colombia) D 21 5 R10630 B.1.617.2 (Delta) D 42 6R10884 B.1.617.2.v2 (Delta) 7 R10801 AY.1 8 R10802 AY.2 9 R11036 AY.4.2

Determination VNTs with homologous and heterologous variants areperformed as described in Example 2 and Example 7. For T-cell analysissplenocytes are isolated on day 42. T-cell analysis by Intracellularcytokine staining (ICS) is performed as described in Example 8. Furtherconstructs encoding new upcoming variants can be tested in a similarway.

Example 9B Vaccination of Rats with mRNA Vaccines Encoding SARS-CoV-2Variants (Prophetic)

Within this study, it can be determined if vaccination with mRNAvaccines encoding SARS-CoV-2 variants induces immunogenicity withcross-neutralizing capacity.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized omicron variantSARS-CoV-2 S (S_stab) are prepared as described in Example 1.2 (RNA invitro transcription). HPLC purified mRNA is formulated with LNPsaccording to Example 1.4 prior to use in in vivo.

Immunization:

Wistar rats are injected intramuscularly (i.m.) with mRNA vaccinecompositions and doses as indicated in Table 14B. As a negative control,one group of rats is vaccinated with buffer (group 1). All animals arevaccinated on day 0 and day 21. Blood samples are collected on day 0,day 14, day 21, and day 42 for the determination of antibody titers.

TABLE 14B Vaccination regimen mRNA Immunisation Serum Group Animals mRNADose schedule isolation 1 Wistar Neg. control: 0.9% NaCl — D 0 D 0Female D 21 D 14 N = 6 D 21 2 Wistar CV2CoV.529 (omicron variant S) natNt 2 μg D 42 3 Female CV2CoV.529 (omicron variant S) nat Nt 8 μg 4 N = 8CV2CoV.529 (omicron variant S) nat Nt 20 μg 5 CV2CoV.529 (omicronvariant S) ψ 2 μg 6 CV2CoV.529 (omicron variant S) ψ 8 μg 7 CV2CoV.529(omicron variant S) ψ 20 μg 8 CV2CoV.529 (omicron variant S) m1ψ 2 μg 9CV2CoV.529 (omicron variant S) m1ψ 8 μg 10 CV2CoV.529 (omicron variantS) m1ψ 20 μg CV2CoV.529 is shown e.g.as R11175, R11176, R11177**,R11178** in Table 4.

Determination VNTs with homologous and heterologous SARS-CoV-2 variantsare performed as described in Example 2. For T-cell analysis splenocytesare isolated on day 42. T-cell analysis by Intracellular cytokinestaining (ICS) is performed as described in Example 8. Furtherconstructs encoding new upcoming variants can be tested in a similarway.

Example 10 Extended Multivalency Vaccination Study: Immunogenicity of aBivalent CV2CoV and CV2CoV.351 Vaccine upon i.m. Administration inWistar Rats

The objective of this study is to assess boost response after CVnCoVvaccination upon a third vaccination with multivalent CV2CoV/CV2CoV.351.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (CV2CoV-mRNA based SARS-CoV-2 vaccineencoding for full length, pre-fusion stabilized ancestral SARS-CoV-2 Sand CV2CoV.351-mRNA based SARS-CoV-2 vaccine encoding for full length,pre-fusion stabilized SARS-CoV-2 B.1.351 S) were prepared as describedin Example 1.2 (RNA in vitro transcription). HPLC purified mRNA wasformulated with LNPs according to Example 1.4 and Example 1.5(separately mixed or formulated for bivalent mRNA vaccines) prior to usein in vivo vaccination experiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 14. Buffer vaccinated animals served asa negative control (group C). All animals were vaccinated on week 0,week 3 (day 21) and on week 15 (day 105). Blood samples were collectedon day 0, day 14, day 21, day 42, day 77, day 105, day 119 and day 133for the determination of antibody titers.

TABLE 15 Vaccination regimen (Example 10) 1st 2nd 3rd vaccinationvaccination vaccination Serum Group Animals week 0 week 3 week 15isolation A N = 4 NaCl NaCl NaCl D 0 (18 h), B Wistar CVnCoV 16 μgCVnCoV 16 μg CV2CoV + Week 2 (D 14) Female CV2CoV.351 8 μg Week 3 (D 21)N = 6 (mixed 2 LNPs) Week 6 (D 42) C CV2CoV 8 μg CV2CoV 8 μg CV2CoV +Week 11 (D 77) CV2CoV.351 8 μg Week 15 (D 105) (mixed 2 LNPs) Week 17 (D119) Week 19 (D 133) CVnCoV is shown as R9515, CV2CoV as R9709 andCV2CoV.351 as R10384 in Table 4.

Determination of VNTs were performed as described in Example 2.

Results:

As shown in FIG. 11A CVnCoV and CV2CoV induced robust VNTs againstancestral SARS-CoV-2 upon two vaccinations in rats. Titers remainedreadily detectable until boosting on day 105 with a small decrease oftiters measured over time for CVnCoV.

Bivalent CV2CoV+CV2CoV.351 vaccine composition showed high boostingcapacity on the background of CVnCoV prime vaccination: VNTs againstancestral SARS-CoV-2 were significantly increased by a factor of 30(d105 vs d119) and a factor of 45 (d105 vs d133) upon CV2CoV+CV2CoV.351boosting (group B). On the background of CV2CoV prime vaccination thebivalent CV2CoV+CV2CoV.351 vaccine composition showed further boostingcapacity of already high VNTs on day 105.

As shown in FIG. 11B CVnCoV induced robust VNTs against SARS-CoV-2B.1.351 upon two vaccinations in rats. Titers directed against B.1.351were overall lower than titers against ancestral virus. Titers remainedreadily detectable until boosting on day 105 with a small decrease ofVNTs measured over time for CVnCoV and CV2CoV vaccinated animals.Bivalent CV2CoV+CV2CoV.351 vaccine composition showed high boostingcapacity on the background of CVnCoV prime vaccination: VNTs againstSARS-CoV-2 B.1.351 were significantly increased by a factor of 19 (d105vs d119) and a factor of 75 (d105 vs d133) upon CV2CoV+CV2CoV.351boosting. On the background of CV2CoV prime vaccination the bivalentCV2CoV+CV2CoV.351 vaccine composition showed further boosting capacityof already high VNTs against SARS-CoV-2 B.1.351 on day 105.

While neutralizing titers detected against B.1.351 remained lower thantiters induced against ancestral SARS-CoV-2 until day 105 of theexperiment, VNTs against both viruses measured upon boosting withCV2CoV+CV2CoV.351 vaccine composition reached comparable levels by day133 (FIG. 11A versus FIG. 11B).

As shown in FIGS. 11-C-11F robust and high VNTs were induced on day 119in both groups B and C not only against ancestral and B.1.351SARS-CoV-2, but also against B.1.1.7 and P.1 SARS-CoV-2 variants (FIG.11C: ancestral, FIG. 11D: B.1.351, FIG. 11E: B.1.1.7, FIG. 11F: P.1).

To conclude, boosting with bivalent CV2CoV+CV2CoV.351 vaccinecomposition three months post two prime vaccinations with CVnCoV orCV2CoV induced a significant increase of VNTs against ancestralSARS-CoV-2 as well as SARS-CoV-2 B.1.351, and elicited high levels ofVNTs against B.1.1.7 and P.1 variants.

Example 11 Vaccination of Rats with mRNA Encoding SARS-CoV-2 VariantB1.617.2 S_Stab Antigen

Preparation of LNP Formulated mRNA Vaccine:

mRNA constructs encoding stabilized Spike (S_stab) of delta variant(B1.617.2) were prepared as described in Example 1.2 (RNA in vitrotranscription). As indicated in Tables 4 and 11, in some constructs,uridine was replaced Pseudouridine (ψ) or by 1-Methylpseudouridinie(m1ψ). HPLC purified mRNA was formulated with LNPs according to Example1.4 prior to use in in vivo vaccination experiments.

Immunization:

Wistar rats (n=8) were injected intramuscularly (i.m.) with mRNA vaccinecompositions and doses as indicated in Table 13. As a negative control,one group of rats was vaccinated with buffer (group 1, n=6). All animalswere vaccinated on day 0 and day 21. Blood samples were collected on day21 (post prime) and 42 (post boost) for the determination of antibodytiters.

TABLE 16 Vaccination regimen (Example 11): SEQ ID SEQ ID mRNA CDS NO:NO: Group Vaccine composition ID opt. Protein RNA Dose 1 buffer — — — —2 CV2CoV.617.2 (delta variant S) R10630 opt1 27095 27394 2 μg 3CV2CoV.617.2 (delta variant S) R10630 opt1 27095 27394 8 μg 4CV2CoV.617.2 (delta variant S) R10630 opt1 27095 27394 20 μg 5CVCoV.617.2 w/o hsl (delta variant S) R10824 opt1 27095 27532 2 μg 6CVCoV.617.2 w/o hsl (delta variant S) R10824 opt1 27095 27532 8 μg 7CVCoV.617.2 w/o hsl (delta variant S) R10824 opt1 27095 27532 20 μg 8CV2CoV.617.2 (delta variant S) ψ R10827 opt1 27095 28762 2 μg 9CV2CoV.617.2 (delta variant S) ψ R10827 opt1 27095 28762 8 μg 10CV2CoV.617.2 (delta variant S) ψ R10827 opt1 27095 28762 20 μg 11CV2CoV.617.2 (delta variant S) m1ψ R10828 opt1 27095 28762 2 μg 12CV2CoV.617.2 (delta variant S) m1ψ R10828 opt1 27095 28762 8 μg 13CV2CoV.617.2 (delta variant S) m1ψ R10828 opt1 27095 28762 20 μgCV2CoV.617.2 is shown as R10630, CVCoV.617.2 w/o hsl as R10824,CV2CoV.617.2 ψ as R10827 and CV2CoV.617.2 m1ψ as R10828 in Table 4. ForR10827 uridine was replaced by ψ (pseudouridinie) and for R10828 uridinewas replaced by m1ψ (1-methylpseudouridinie).

Determination of total IgG spike-binding antibody titers using ELISA anddetermination of VNTs were performed as described in Example 2.Recombinant SARS-CoV-2 spike B.1.617.2 RBD protein (L452R, T478K, deltavariant) was used for ELISA IgG determination. (for VNTs against deltaB.1.617.2 the following strain was used: Lineage: Delta—B.1.617.2,Strain:hCoV-19/France/IDF-APHP-HEGP-20-23-2131905084/2021|EPI_ISL_2029113|2021-04-27(T19R E156G d157F d158R L452R T478K D614G P681R D950N).

Results:

As shown in FIGS. 12A and B vaccination with different mRNA formatsencoding full length S stabilized protein (delta variant B1.617.2)formulated in LNPs induced in rats significant levels of spike-bindingantibody titers on day 14 and day 42 using doses of 2 μg, 8 μg, and 20μg. The second vaccination led to a further increase of antibody titers.Robust VNTs were induced against SARS-CoV-2 variant B1.617.2 in a dosedependent manner for the 2, 8, and 20 μg groups that increasing overtime. VNTs were detectable as early as d14 post first injection for allgroups including the 2 μg dose (FIG. 12A, day 14), FIG. 12B (day 21),and FIG. 12C (day 42). Robust heterologous VNTs against ancestralSARS-CoV-2 (FIG. 12F), against SARS-CoV-2 variant B.1.351 (FIG. 12G),and against SARS-CoV-2 variant P.1 (FIG. 12H) were induced too.

The results demonstrate that the introduction of natural nucleotides(groups 2-7) or chemically modified nucleotides ((ψ (pseudouridinie,groups 8-10) or m1ψ (1-methylpseudouridinie, groups 11-13)) into themRNA constructs induce comparable levels of VNTs against differentvariants on day 42, with a trend to improved VNTs by using chemicallymodified nucleotides (groups 8-13).

Example 12 Multivalency Study in Rats: Immunogenicity of a BivalentVaccine Compositions upon i.m. Administration in Wistar Rats

In this study, humoral immunogenicity induced by differentLNP-formulated bivalent mRNA vaccine compositions was evaluated inWistar rats.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs (mRNA based SARS-CoV-2 encoding for fulllength, pre-fusion stabilized variant SARS-CoV-2 S) were prepared asdescribed in Example 1.2 (RNA in vitro transcription). HPLC purifiedmRNA was formulated with LNPs according to Example 1.4 and Example 1.5(separately mixed or formulated for bivalent mRNA vaccines) prior to usein in vivo vaccination experiments.

Immunization:

Rats were injected intramuscularly (i.m.) with bivalent mRNA vaccinecompositions and doses as indicated in Table 6. Buffer vaccinatedanimals served as a negative control (group 1). All animals werevaccinated on day 0 and day 21. Blood samples were collected on day 14,day 21 and day 42 for the determination of humoral immune responses.

TABLE 17 Vaccination regimen (Example 12) SEQ ID SEQ ID mRNA NO: NO:Immunisation Serum Group Animals Vaccine composition ID Protein RNA Doseschedule isolation 1 N = 4 Neg control — — — D 0 D 1 (buffer) D 21 D 142 Wistar CV2CoV.617.2 R10630 27095 27394 8 μg D 21 3 Female CV2CoV.617.2m1ψ R10828 27095 28762 8 μg D 42 4 N = 6 CV2CoV + R9709 + 10 + 149 + 8μg CV2CoV.617.2 R10630 27095 27394 5 CV2CoV m1ψ. + R10159 + 10 + 149 + 8μg CV2CoV.617.2 m1ψ R10828 27095 28762 6 CV2CoV.351 + R10384 + 22961 +23531 + 8 μg CV2CoV.617.2 R10630 27095 27394 7 CV2CoV D614G + R10166 +22738 + 22792 + 8 μg CV2CoV.617.2 R10630 27095 27394 8 CV2CoV D614Gm1ψ + R10813 + 22738 + 28737 + 8 μg CV2CoV.617.2 m1ψ R10828 27095 28762For some constructs, uridine was replaced by m1ψ(1-methylpseudouridinie): R10828, R10159, R10813. Determination of totalIgG spike-binding antibody titers using ELISA was performed as describedin Example 2. Recombinant Spike RBD protein of ancestral SARS-CoV-2, RBDof variant B.1.617.2 (L452R, T478K: delta), or RBD of B.1.351 (K417N,E484K, N501Y: beta) was used for coating.

Results:

Vaccination with bivalent vaccine compositions comprising different mRNAformats comprising natural or chemically modified nucleotides (m1ψ(1-methylpseudouridinie)) encoding full length stabilized Spike proteinof different variants (for more details see Table 17) formulated in LNPsinduced in rats robust and high levels of spike-binding antibody titerson day 14. FIG. 13 demonstrates homologous as well as heterologousresponses. (FIG. 13A: ancestral SARS-CoV-2 RBD; FIG. 13B: B.1.617.2 RBD(L452R, T478K, delta); FIG. 13C: B.1.351 RBD (K417N, E484K, N501Y,beta). The constructs with modified nucleotides induce higher total IgGtiter than constructs with natural nucleotides, for homologous as wellas for heterologous responses.

Example 13 Challenge Study in k18-hACE2 Mice with SARS-CoV-2 B.1.351 andB.1.617.2

Generally, mice are not susceptible to infection with SARS-CoV-2, but agenetically engineered mouse model has been developed that expresses thehuman receptor ACE2 (hACE2), required for entry of the virus into thehost cell under the K18 promoter. The model was originally developed toinvestigate the causative agent of SARS (SARS-CoV) (MCCRAY, Paul B., etal. Lethal infection of K18-hACE2 mice infected with severe acuterespiratory syndrome coronavirus. Journal of virology, 2007, 81. Jg.,Nr. 2, S. 813-821) but is now also used as a suitable small animal modelfor COVID-19. Previously, hACE2 mice have been shown to be susceptibleto SARS-CoV-2 and to exhibit a disease course with weight loss,pulmonary pathology, and symptoms similar to those in humans (e.g. BAO,Linlin, et al. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.Nature, 2020, 583. Jg., Nr. 7818, S. 830-833, or YINDA, Claude Kwe, etal. K18-hACE2 mice develop respiratory disease resembling severeCOVID-19. PLoS pathogens, 2021, 17. Jg., Nr. 1, S. e1009195; DE ALWIS,Ruklanthi M., et al. A Single Dose of Self-Transcribing and ReplicatingRNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity InMice. BioRxiv, 2020.). In principle, the K18-hACE2 mouse is suitable forvaccine studies to investigate the prevention of infection withSARS-CoV-2 or SARS-CoV-2 variants or the reduction of viral load, and atthe same time to investigate the correlates and causes of a protectiveeffect of an mRNA vaccine against COVID-19 with well-establishedimmunological methods, which are generally available for mouse models.

The present example shows that SARS-CoV-2 variant S mRNA vaccines areable to protect K18-hACE2 mice from SARS-CoV-2 viral challenge, whichcan be shown e.g. by measuring the viral loads of infected animals, bymonitoring the disease progression with weight loss, pulmonary pathologyand other symptoms, or by histopathology and survival.

Preparation of LNP Formulated mRNA Vaccine:

mRNA constructs for SARS-CoV-2 vaccine were prepared as described inExample 1.2 (RNA in vitro transcription). HPLC purified mRNA wasformulated with LNPs according to Example 1.4 and Example 1.5(separately mixed or formulated for bivalent mRNA vaccines) prior to usein in vivo vaccination experiments.

Immunization and Challenge:

K18-hACE2 transgenic mice (female, n=2×10) were injected intramuscularly(i.m.) with mRNA vaccine compositions as indicated in Table 18 (group1-4). As a negative control, one group of mice was treated with buffer(group 5). The animals were vaccinated on day 0 and day 28 withindicated doses at a volume of 20 μl. Blood samples were collected onday 0, day 28 (post prime), day 56 (post boost) and day 66 (postchallenge) for the determination of antibody titers. The animals werechallenged/infected i.n. with SARS-CoV-2 virus ((10^(4.375) TCID₅₀ permouse SARS-CoV-2 B.1.351 and 10^(4.375) TCID₅₀ SARS-CoV-2 B.1.617.2,calculated from back titration of the original material) on day 56 andmonitored for 10 days for changes in body weight, general health andsurvival, which indicates protection from challenge. Additionalparameters of protection include reduced viral loads in lungs and otherorgans and reduced pathology of the lung. RNA extraction and RT-qPCR andsgRNA RT-PCR was performed as described in Hoffmann et al 2021(Hoffmann, D., Corleis, B., Rauch, S. et al. CVnCoV and CV2CoV protecthuman ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351SARS-CoV-2. Nat Commun 12, 4048 (2021)).

TABLE 18 Vaccination regimen (Example 13): 5′-UTR/ SEQ ID SEQ ID mRNA3′-UTR; NO: NO: Group Vaccine composition ID UTR Design 3′-end ProteinRNA Dose 1 CV2CoV R9709 HSD17B4/ hSL-A100 10 149 0.5 μg PSMB3; a-1 2CV2CoV.351 R10384 HSD17B4/ hSL-A100 22961 23531 0.5 μg PSMB3; a-1 3CV2CoV.617.2 R10630 HSD17B4/ hSL-A100 27095 27394 0.5 μg PSMB3; a-1 4CV2CoV.351 + R10384 + HSD17B4/ hSL-A100 22961 + 23531 + 0.25 μg +CV2CoV.617.2 R10630 PSMB3; a-1 27095 27394 0.25 μg 5 NaCl buffer

RBD Antibody Enzyme-Linked Immunosorbent Assay (ELISA)

Sera were analyzed using an indirect multi-species ELISA based on theRBD (ancestral) of SARS-CoV-2. For this, ELISA plates (Greiner Bio-OneGmbH) were coated with 100 ng/well the RBD overnight at 4° C. in 0.1 Mcarbonate buffer (1.59 g Na₂CO₃ and 2.93 g NaHCO₃, ad. 1 L aqua dest.,pH9.6) or were treated with the coating buffer only. Afterwards, theplates were blocked for 1 h at 37° C. using 5% skim milk in PBS. Serawere pre-diluted 1/100 in TBS-Tween (TBST) and incubated on the coatedand uncoated wells for 1 h at RT. A multi-species conjugate (SBVMILK;obtained from ID Screen® Schmallenberg virus Milk Indirect ELISA; IDvet)was diluted 1/80 and then added for 1 h at RT. Following the addition oftetramethylbenzidine substrate (IDEXX), the ELISA readings were taken ata wavelength of 450 nm on a Tecan Spectra Mini instrument (Tecan GroupLtd.). Between each step, the plates were washed three times with TBST.The absorbance was calculated by subtracting the optical densitymeasured on the uncoated wells from the values obtained from theprotein-coated wells for the respective sample. Of note, the ELISAdetermines relative abundance of anti-RBD Ig levels and therefore doesnot allow a direct comparison between different studies.

Virus Neutralization Test (VNT)

Sera were pre-diluted 1/16 or 1/32—with DMEM in a 96 well deep wellmaster plate. In three replicate studies, 100 μl of this pre-dilutedsamples were transferred into a 96 well plate. A log 2 dilution wasconducted by passaging 50 μl of the serum dilution in 50 μl DMEM,leaving 50 μl of sera dilution in each well. Subsequently 50 μl of therespective SARS-CoV-2 (B.1.351 or B.1.617.2) virus dilution (100TCID50/well) was added to each well and incubated for 1 hour at 37° C.Lastly, 100 μl of trypsinated VeroE6 cells (cells of one confluent TC175flask per 100 ml) in DMEM with 1% penicillin/streptomycinsupplementation was added to each well. After 72 hours incubation at 37°C., the wells were evaluated by light microscopy. A serum dilution wascounted as neutralizing in the case no specific CPE was visible. Thevirus titer was confirmed by virus titration, positive and negativeserum samples were included.

Results:

Vaccine efficacy was tested by challenging mice with either SARS-CoV-2variant B.1.351 or SARS-CoV-2 variant B.1.627.2. As shown in FIG. 14,mice of all vaccination groups (group1-4) benefit from vaccination withcomposition comprising mRNA encoding SARS-CoV-2 ancestral or variantSpike proteins. FIGS. 14A and B demonstrate survival of challenged micein days post infection/challenge (FIG. 14A: challenge with B.1.351, FIG.14B: challenge with B.1.617.2). Vaccination with all tested mRNAvaccines resulted in complete protection of mice (100% survival) againstboth tested SARS-CoV-2 variants, irrespective of encoded spike variant(group 1: ancestral, group 2: B.1.351, group 3 B.1.617.2, group 4B.1.351+3 B.1.617.2). FIGS. 14C and D demonstrate the percentage bodyweight changes in days post infection/challenge (FIG. 14C: challengewith B.1.351, FIG. 14D: challenge with B.1.617.2, mean percentage bodyweight). Mice of all vaccination groups (group 1-4) did not showsignificant weight loss.

To investigate whether the vaccination prevented productive infection ordissemination of replicating SARS-CoV-2, oral swabs were taken on day 4post infection to monitor viral RNA load in saliva. In the sham group,8/9 or 6/9 samples were positive for viral genome after infection withSARS-CoV-2 variant B.1.351 or SARS-CoV-2 B.1.617.2, respectively (FIG.14E: B.1.351 challenge group, FIG. 14F: B.1.617.2 challenge group). Incontrast, after mRNA vaccination, no viral genomes were detected in oralswabs of either challenge groups irrespective of the vaccine group (onlyin one mouse of CV2CoV vaccination group viral genomes were detectedFIG. 14E, group 1). To further explore the prevention of viralreplication following challenge, viral load in the upper respiratorytract (URT) (conchae) and the lower respiratory tract (LRT) (lung), aswell as in the central nervous system (brain, cerebellum/cerebrum) wasanalyzed 10 days post infection. In mice challenged with SARS-CoV-2B.1.351, reduction of detectable viral replication was observed in allvaccination groups compared to mice not vaccinated (group 5) in the URT(FIG. 14G). This effect was more prominent for mice challenged withSARS-CoV-2 variant B.1.617.2 (FIG. 14H). Mice vaccinated with LNP-mRNAcoding for SARS-CoV-2 spike variant B.1.617.2 (group 3) showed noreplication in conchae after homologous virus challenge (FIG. 14H). Noanimal was positive at a low level for SARS-CoV-2 RNA in the LRT,indicating protection from infection by SARS-CoV-2 variant B.1.351 andSARS-CoV-2 variant B.1.617.2 in all groups (FIGS. 14I and J,respectively). For the brain, similar results were achieved (FIGS. 14Kand L for cerebellum, FIGS. 14M and N for Cerebrum (for challenge groupB.1.351: FIGS. 14K and M, for B.1.617.2: FIGS. 14L and N).

The bivalent vaccine (group 4) induced protection against both virusvariants (B.1.351 and B.1.617.2) comparable to the monovalent vaccines(groups 1-3) despite using lower doses of each vaccine.

Sera from all vaccinated mice collected on day 28 (only tested forchallenge group B.1.351) and day 56 (for both groups) showed a stronginduction of anti-RBD total immunoglobulins (Ig), irrespective of whichvariant spike the mRNA was coding (FIG. 14O: challenge group B.1.351,FIG. 14P: challenge group B.1.617.2). The strong induction of anti-RBDantibodies in the mRNA vaccine groups was reflected by high virusneutralization titers (VNT) (FIG. 14Q: post-challenge group B.1.351,FIG. 14R: pre-challenge group B.1.617.2, FIG. 14S: post-challenge groupB.1.617.2). Overall, the tested mRNA vaccines induced robust antibodyresponses in a prime-boost regime, capable of efficiently neutralizingboth SARS-CoV-2 variants B.1.351 and SARS-CoV-2 B.1.617.2 in vitro.

Example 14 Challenge Study of Hamsters Vaccinated with CV2CoV orCV2CoV.351

The protective efficacy of LNP-mRNA encoding variant B.1.351 S_stabformulated in LNPs (CV2CoV.351) was addressed in Syrian hamsters. Thismodel represents mild to moderate human lung disease pathology and isone of the recognized and accepted models to investigate human-relevantimmunogenicity and pathogenesis (Muñoz-Fontela et al, PMID 32967005).Hamsters are susceptible to wild-type SARS-CoV-2 infection, resulting inhigh levels of virus replication and histopathological changes in viraltarget organs.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs were prepared as described in Example 1(RNA in vitro transcription). HPLC purified mRNA was formulated withLNPs according to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization and Challenge:

Syrian golden hamsters (n=9/group) were injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 19. Asnegative controls, one group of hamsters was not treated and mockinfected (with buffer) (group A), another group was injected with NaClas a buffer control. The animals were challenged intranasally under ashort-term inhalation anaesthesia with 70 μl of SARS-CoV-2 variantB.1.351 using 10^(5.25) TCID50 per hamster (calculated from backtitration of the original material) at day 56. For the days afterinfection, viral shedding was monitored in addition to a daily physicalexamination and body weighing routine. To evaluate viral shedding, nasalwashes were individually collected from each hamster under a short-termisoflurane anaesthesia. Blood samples were collected on day 28 (postprime), day 55 (post boost) and day 60 (post challenge infection) forthe determination of antibody titers.

TABLE 19 Vaccination regimen (Example 14): SEQ ID SEQ ID mRNA NO: NO:Group Vaccine composition ID dose vaccination Protein RNA 1 NaCl — — d0, d 28 2 CV2CoV.351 R10384 1 μg d 0, d 28 22961 23531 3 CV2CoV.351R10384 4 μg d 0, d 28 22961 23531 4 CV2CoV.351 R10384 12 μg  d 0, d 2822961 23531

Antibody Analysis

Blood samples were taken at days 0, 28, 55, and 60 for the determinationof total IgG antibodies via ELISA. Plates were coated with 1 μg/ml ofSARS-CoV-2 S ancestral RBD for 4-5 h at 37° C. Plates were blockedovernight in 10% milk, washed and incubated with serum for 2 h at roomtemperature. For detection, hamster sera were incubated with biotin goatanti-hamster (Syrian) IgG antibody (BioLegend, Cat: 405601) followed byincubation with HRP-Streptavidin (BD, Cat: 554066). Detection ofspecific signals was performed in a BioTek SynergyHTX plate reader, withexcitation 530/25, emission detection 590/35 and a sensitivity of 45.

Virus neutralizing antibody titers (VNT) of hamster serum samples wereanalyzed as described in Example 13 by using only SARS-CoV-2 virusvariant B.1.351.

Viral Load in the Respiratory Tract

RNA was isolated from nasal wash over time and from lung tissue samples(cranial, medial, caudal) 4 days post challenge infection. RNAextraction followed by detection of subgenomic RNA (sgRNA) by RT-qPCRwas performed as described in Hoffmann et al 2021 (Hoffmann, D.,Corleis, B., Rauch, S. et al. CVnCoV and CV2CoV protect human ACE2transgenic mice from ancestral B BavPatl and emerging B.1.351SARS-CoV-2. Nat Commun 12, 4048 (2021)).

Results

Vaccine efficacy was tested by challenging hamsters with 10^(5.25)TCID50 dose/hampster of SARS-CoV-2 variant B.1.351. FIG. 15Ademonstrates the percentage body weight changes in days post challenge.Mice of all vaccination groups (group 2-4) did not show significantweight loss. The weight of non-treated mice decreased over time down to90% body weight (group 1).

To investigate whether the vaccination prevented productive infection ofSARS-CoV-2, nasal washes were analyzed on days 2, 4, 8, and 12 postinfection to monitor viral RNA load of infected animal. In vaccinatedgroups (groups 2-4), the viral load determined by detecting sgRNA, wasslightly reduced compared to the untreated control (group 1) (FIG. 15B).More prominent, significant reduced viral genomes (by detecting sgRNA)were detected in the lower respiratory tract (LRT) (of cranial, medial,caudal lung lobes) after mRNA vaccination. Only one animal was positiveat a low level for SARS-CoV-2 B.1.351 RNA in the 12 μg CV2CoV.351vaccine group (group 4), indicating protection from infection bySARS-CoV-2 variant B.1.351 (FIG. 15C).

Sera from mice collected on day 28, day 55 and day 60 showed a stronginduction of anti-RBD total immunoglobulins (Ig) (FIG. 15D). Thisinduction of anti-RBD antibodies in the mRNA vaccine groups wasreflected by robust virus neutralization titers (VNT) (see FIG. 15E(open symbol pre-challenge (day 55), filled symbols post-challenge onday 60). Overall, the tested mRNA vaccines induced robust antibodyresponses in a prime-boost regime, capable of efficiently neutralizingSARS-CoV-2 variant B.1.351.

Example 15 Prophetic

To demonstrate safety and efficiency of the mRNA vaccine composition(s),a clinical trial (phase I) is initiated. In the clinical trial, a cohortof human volunteers is intramuscularly injected for at least two times(e.g. day 0 and day 28) with mRNA encoding variant SARS-CoV-2 spikeprotein formulated in LNPs according to the invention. In order toassess the safety profile of the vaccine compositions according to theinvention, subjects are monitored after administration (vital signs,vaccination site tolerability assessments, hematologic analysis). Theefficacy of the immunization is analyzed by determination of virusneutralizing titers (VNT) in sera from vaccinated subjects. Bloodsamples are collected on day 0 as baseline and after completedvaccination. Sera are analyzed for virus neutralizing antibodies.

1. A composition comprising (a) an mRNA comprising: (i) at least onecoding sequence encoding a SARS-CoV-2 spike protein (S) at least 90%identical to SEQ ID NO: 10 that is a pre-fusion stabilized spike protein(S_stab) comprising K986P and V987P stabilizing mutations and 10 or moreof the following substitutions relative to SEQ ID NO: 10: A67V, H69del,V70del, T95I, G142D, V143del, Y144del, Y145del, N211del, L212I,ins214EPE, G339D, 5371L, S373P, S375F, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K and L981F, and (ii) at least one heterologousuntranslated region (UTR); and (b) at least one pharmaceuticallyacceptable carrier, wherein the mRNA is complexed or associated with alipid nanoparticle (LNP) and wherein the LNP comprises: (i) at least onecationic lipid; (ii) at least one neutral lipid; (iii) at least onesteroid or steroid analogue; and (iv) at least one PEG-lipid, wherein(i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25%neutral lipid, 25-55% sterol, and 0.5-5% PEG-lipid.
 2. A compositioncomprising (a) an mRNA comprising: (i) at least one coding sequenceencoding a SARS-CoV-2 spike protein (S) at least 95% identical to SEQ IDNO: 10 and is a pre-fusion stabilized spike protein (S_stab) comprisingK986P and V987P stabilizing mutations and T19R, F157del, R158del, L452R,T478K, D614G, P681R and D950N amino acid substitutions relative to SEQID NO: 10, and (ii) at least one heterologous untranslated region (UTR);and (b) at least one pharmaceutically acceptable carrier, wherein themRNA is complexed or associated with a lipid nanoparticle (LNP) andwherein the LNP comprises: (i) at least one cationic lipid; (ii) atleast one neutral lipid; (iii) at least one steroid or steroid analogue;and (iv) at least one PEG-lipid, wherein (i) to (iv) are in a molarratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55%sterol, and 0.5-15% PEG-lipid.
 3. A composition comprising (a) an mRNAcomprising: (i) at least one coding sequence encoding a SARS-CoV-2 spikeprotein (S) at least 95% identical to SEQ ID NO: 10 that is a pre-fusionstabilized spike protein (S_stab) comprising K986P and V987P stabilizingmutations and 10 or more of the following substitutions relative to SEQID NO: 10: A67V, H69del, V70del, T95I, G142D, V143del, Y144del, Y145del,N211del, L212I, ins214EPE, G339D, 5371L, S373P, S375F, S477N, T478K,E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,P681H, N764K, D796Y, N856K, Q954H, N969K and L981F, and (ii) at leastone heterologous untranslated region (UTR); (b) an mRNA comprising: (i)at least one coding sequence encoding a SARS-CoV-2 spike protein (S) atleast 95% identical to SEQ ID NO: 10 and is a pre-fusion stabilizedspike protein (S_stab) comprising K986P and V987P stabilizing mutationsand T19R, F157del, R158del, L452R, T478K, D614G, P681R and D950N aminoacid substitutions relative to SEQ ID NO: 10, and (ii) at least oneheterologous untranslated region (UTR); and (c) at least onepharmaceutically acceptable carrier, wherein the mRNA is complexed orassociated with a lipid nanoparticle (LNP) and wherein the LNPcomprises: (i) at least one cationic lipid; (ii) at least one neutrallipid; (iii) at least one steroid or steroid analogue; and (iv) at leastone PEG-lipid, wherein (i) to (iv) are in a molar ratio of about 20-60%cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-5%PEG-lipid.
 4. The composition of claim 1, wherein the mRNA comprises atleast one poly(A) sequence comprising 30 to 200 adenosine nucleotidesand a 5′-cap structure.
 5. The composition of claim 1, wherein the atleast one coding sequence of the mRNA has a G/C content of at leastabout 50%.
 6. The composition of claim 3, wherein the at least onecoding sequence of the mRNA has a G/C content of at least about 55%. 7.The composition of claim 1, wherein the mRNA comprises a sequence atleast 90% identical to SEQ ID NO:
 28590. 8. The composition of claim 1,wherein the at least one heterologous UTR is selected from at least oneheterologous 5′-UTR and/or at least one heterologous 3′-UTR.
 9. Thecomposition of claim 8, wherein the at least one heterologous 3′-UTRcomprises or consists of a nucleic acid sequence derived from a 3′-UTRof a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS,NDUFA1 and RPS9; or wherein the at least one heterologous 5′-UTRcomprises or consists of a nucleic acid sequence derived from a 5′-UTRof a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4,NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2.
 10. The composition of claim 1,wherein the mRNA comprises at least one histone stem-loop.
 11. Thecomposition of claim 1, wherein the mRNA comprises a nucleotide analogsubstitution.
 12. The composition of claim 11, wherein the mRNAcomprises a pseudouridine or 1-methylpseudouridine substitution at auridine position.
 13. The composition of claim 1, wherein the mRNAcomprises a 1-methylpseudouridine substitution at a uridine position.14. The composition of claim 1, wherein the mRNA has an RNA integrity ofat least about 50%.
 15. The composition of claim 1, wherein the mRNA isa purified mRNA that has been purified by RP-HPLC and/or TFF.
 16. Thecomposition of claim 15, wherein the mRNA is a purified mRNA that hasbeen purified by RP-HPLC and/or TFF and comprises about 5%, 10%, or 20%less double stranded RNA side products as compared to an RNA that hasnot been purified with RP-HPLC and/or TFF.
 17. The composition of claim1, wherein the LNP comprises a cationic lipid according to formula III:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: L¹ or L² is each independently —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)C(═O)—,—C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—,preferably L¹ or L² is —O(C═O)— or —(C═O)O—; G¹ and G² are eachindependently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ isC₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, or C₃-C₈cycloalkenylene; R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are eachindependently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H orC₁-C₆ alkyl; and x is 0, 1 or
 2. 18. The composition of claim 17,wherein the LNP comprises a cationic lipid according to formula III-3:


19. The composition of claim 17, wherein the LNP comprises a PEG lipidaccording to formula IVa:


20. The composition of claim 18, wherein the LNP comprises: (i) at leastone cationic lipid according to formula (III-3); (ii) at least oneneutral lipid comprising 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC); (iii) at least one sterol comprising cholesterol; and (iv) atleast one PEG-lipid according to formula (IVa), wherein (i) to (iv) arein a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid,25-55% sterol, and 0.5-15% PEG-lipid.
 21. The composition of claim 20,wherein the mRNA comprises a sequence at least 90% identical to SEQ IDNO:
 163. 22. The composition of claim 1, further comprising alyoprotectant.
 23. The composition of claim 22, wherein thelyoprotectant comprises sucrose.
 24. The composition of claim 1, whereinthe composition comprises less than about 20% free mRNA.
 25. Thecomposition of claim 1, wherein the LNP have a mean diameter of fromabout 60 nm to 200 nm.
 26. The composition of claim 1, wherein thecomposition has a lipid to RNA molar ratio (N/P ratio) of from about 2to about
 12. 27. The composition of claim 15, wherein the compositionhas a lipid to RNA molar ratio (N/P ratio) of from about 2 to about 12.28. A kit comprising the composition according to claim 1 and optionallycomprising a liquid vehicle for solubilising, and, optionally, technicalinstructions providing information on administration and dosage for use.29. A method of treating or preventing a disorder, wherein the methodcomprises applying or administering to a subject in need thereof thecomposition according to claim
 1. 30. A composition comprising a mRNAcomprising: (a) at least one coding sequence encoding a SARS-CoV-2 spikeprotein (S) at least 95% identical to SEQ ID NO: 10 that is a pre-fusionstabilized spike protein (S_stab) comprising K986P and V987P stabilizingmutations and H69del, V70del, S477N, T478K, E484A, N501Y, D614G, P681H,H655Y, Q493R, T95I, A67V and G142D amino acid substitutions relative toSEQ ID NO: 10; (b) at least one heterologous untranslated region (UTR);and (c) at least one pharmaceutically acceptable carrier, wherein themRNA is complexed or associated with a lipid nanoparticle (LNP) andwherein the LNP comprises: (i) at least one cationic lipid; (ii) atleast one neutral lipid; (iii) at least one steroid or steroid analogue;and (iv) at least one PEG-lipid, wherein (i) to (iv) are in a molarratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55%sterol, and 0.5-5% PEG-lipid.